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ONCOLOGY NANOMEDICINE: Study of InteractionsBetween Nanoparticles Activated by External

Electromagnetic Energy Sources and Cancer CellsforEnhancement of the Therapeutic Window

Simon Virginie

To cite this version:Simon Virginie. ONCOLOGY NANOMEDICINE: Study of Interactions Between Nanoparticles Ac-tivated by External Electromagnetic Energy Sources and Cancer Cells forEnhancement of the Ther-apeutic Window. Life Sciences [q-bio]. Université Pierre et Marie Curie - Paris VI, 2009. English.�tel-00831605�

https://tel.archives-ouvertes.fr/tel-00831605

https://hal.archives-ouvertes.fr

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THESE DE DOCTORAT DE

L’UNIVERSITE PARIS VI - PIERRE ET MARIE CURIE

Ecole doctorale ‘Complexité du Vivant’

Spécialité Biologie Cellulaire

Présentée par

Virginie SIMON

Pour obtenir le grade de

DOCTEUR de l’UNIVERSITÉ PIERRE ET MARIE CURIE

Sujet de la thèse :

ONCOLOGY NANOMEDICINE:

Study of Interactions Between Nanoparticles Activated by

External Electromagnetic Energy Sources and Cancer Cells for

Enhancement of the Therapeutic Window

Soutenue le 3 décembre 2009 à Paris devant le jury composé de :

Michel MORANGE Président du Jury Alex DUVAL Directeur de thèse Julie MARILL Co-directeur Véronique ROSILIO Rapporteur Eric DEUTSCH Rapporteur

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“Un coeur né pour la liberté ne se laisse jamais traiter en esclave”

Mozart, L’énlèvement au sérail.

A ma famille

A Tristan

- 4 -

- J’adresse tous mes remerciements

Aux membres de mon jury de thèse. A Michel Morange, docteur en biologie et en

philosophie, professeur de biologie à l’ENS (Ecole Normale Supérieure) et à l’Université

Paris VI, d’avoir accepté d’être le président de ce jury de thèse. A Véronique Rosilio, docteur

en pharmacie, chercheur en physico-chimie des surfaces à Châtenay-Malabry et au docteur

Eric Deutsch, oncologue radiothérapeute à l’IGR (Institut Gustave Roussy, Villejuif) ; tous les

deux rapporteurs de cette thèse ; pour avoir pris le temps d’apporter de nouvelles

interrogations aux nombreuses thématiques soulevées au cours de ce travail. Enfin, à mes

deux directeurs de thèse Alex Duval, généticien et directeur d’équipe appartenant à l’unité

Inserm et Paris VI UMRS 938 « Instabilité des microsatellites et cancer » et à Julie Marill,

docteur en biologie et chercheur à Nanobiotix. Je te remercie Alex de m’avoir accueillie

pendant les premiers mois de ma thèse au sein de l'hôpital Saint-Louis, à la Fondation Jean

Dausset-CEPH. Merci pour tes conseils, ton ouverture d’esprit (je citerai tes passions pour les

voyages, la musique et le cinéma) et d’avoir toujours été compréhensif quant à ma situation

de thésarde du privé.

A Laurent Levy, docteur en physique et CEO de Nanobiotix et à Kader Boussaha, CFO et

COO de Nanobiotix, fondateurs de cette start-up, de m’avoir permis d’effectuer mon stage de

Master II de génétique à l’Université Paris VI et de fin de cycle d’ingénieur de l’UTC

(Université Technologique de Compiègne) à Nanobiotix et de m’avoir accueillie au sein de la

start-up naissante. Je vous remercie également de m’avoir proposé par la suite d’effectuer ma

thèse en convention Cifre et d’avoir pu engager l’approfondissement de la compréhension des

nanoparticules avec les cellules cancéreuses. De février 2006 à octobre 2009, j’ai pu grâce à

vous observer et participer à l’évolution d’une société innovante. Je vous remercie enfin

d’avoir toujours pris le temps de m’expliquer le fonctionnement administratif, juridique et

financier de Nanobiotix m’ouvrant ainsi un peu plus mes connaissances de ce monde

industriel.

A Elsa Borghi, directrice médicale à Nanobiotix et à Agnès Pottier, directrice du département

recherche à Nanobiotix pour m’avoir tant soutenue et poussée dans l’approfondissement des

connaissances à tous les niveaux de mon travail de thèse : rédaction d’articles, rédaction du

manuscrit et préparation de la soutenance orale. Merci infiniment à toutes les deux.

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- Un grand merci

A l’ensemble de l’équipe de Nanobiotix. A Corinne Devaux qui m’a accueillie à mon arrivée

dans les premiers locaux dans le 2ème arrondissement de Paris. A Ping Zhang et Laurence

Maggiorella, chercheurs en biologie, qui travaillent à l’IGR. A Maria Di Marco pour nos

rendez-vous gourmands du midi. A Fazia Chelli présente à la fin de cette aventure. A Brigitte

Congard-Chassol pour ses formations à la qualité en entreprise. A Patricia Said et ses Louises.

A Elodie Menard et sa gentillesse. A Thibault Donnet, mon stagiaire de l’EBI (Ecole de

Biologie Industrielle) que j’ai pu encadrer. Merci pour ton investissement et ta passion de la

recherche.

Sans oublier bien évidemment « l’open space » où j’ai passé deux ans et demi avec Audrey

Darmon, Laurence Poul, Matthieu Germain, Marie-Edith Meyre, Annabelle Rouet, Kelthoum

Piejos et Edouard Thiénot. Je retiendrai nos fous rires et nos soirées. Audrey, quelle chance de

t’avoir rencontrée et d’avoir travaillé ensemble sur la totalité du projet nanoPDT. Tu m’as

aidée techniquement, moralement, intellectuellement et merci pour ton témoignage sur la

vidéo de mon prix Excellencia � Pupule, j’ai également été ravie de travailler pendant près

d’un an à tes côtés et de voir ta volonté de comprendre, d’analyser et d’approfondir tout ce

que tu entreprends. Tous ces moments passés ensemble, les pauses, les repas, les départs, les

pots, les soirées, les télomérases, les parthénogénèses et les « loin sans dire ». Matou et Mémé

qui m’ont permis de connaître un peu mieux le monde des liposomes et des binouzes à moins

que ce soit les steaks... Enfin, Nanabelle et Kette (on va dire ça comme ça !) le binôme de

choc qui traverse les étapes et les difficultés de la production et du développement de

NBTXR3. Nos bavardages me manquent déjà ! Mais je suis confiante quant au fait de rester

en contact avec vous tous. Beaucoup de bonne humeur dans cet « open space », entretenez la.

A l’ensemble de l’équipe d’Alex Duval de m’avoir permis de coécrire un article dans le

bulletin du cancer « Conséquences cliniques et moléculaires de l’instabilité des microsatellites

dans les cancers humains » en 2008. Merci plus particulièrement à Alexandra Chalastanis,

Ann-Sofie Schreurs, Olivier Buhard et Richard Hamelin. Alexandra ma jumelle de thèse,

Ann-So la tête dans les étoiles, Olivier le chasseur d’orages et Richard à la recherche de

peintures murales.

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A Marcel Lahmani, Patrick Boisseau et Philippe Houdy de m’avoir permis de participer à la

rédaction du chapitre 25 “Vers des nanoparticules activables pour le traitement du cancer:

l’exemple de Nanobiotix”, Les nanosciences 3. Nanobiotechnologies et nanobiologie aux

éditions Belin en 2007.

A l’équipe de Paris VI de microscopie électronique à transmission (MET) et à balayage

(MEB) pour m’avoir formée dans le but d’être entièrement autonome à ces techniques

délicates (coupes aux diamants pour le MET).

A l’école doctorale « Complexité du vivant » pour m’avoir permis d’obtenir mon diplôme

d’expérimentation animale de niveau I en suivant la formation appropriée.

Aux « persos » présentes à ma soutenance de thèse qui s’est réalisée en huit clos. Un grand

merci à mes parents qui m’ont toujours soutenue. A Tristan bien évidemment. A Alex, Céline,

Mélanie, Quentin, Hugo ainsi qu’à mes amies de longue date non présentes Anne, Marie et

Fanny. Pensées à ma sœur Nathalie, Manu, mon neveu Maxence et ma nièce Hortense. Enfin,

merci à tous mes autres ami(e)s que je ne nommerai pas par crainte d’en omettre certains.

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ABSTRACT

The understanding of interactions between nanomaterials and biological entities is

fundamental to develop nanoproducts in oncology. NBTXR3 (hafnium oxide inert

nanoparticle) and protoporphyrin IX (Pp IX) nanocarrier (silica nanoparticle encapsulating Pp

IX, the monomer of Photofrin®) are nanoproducts, issued from Nanobiotix platforms, aim to

enlarge the therapeutic window of oncology treatment, using external energy sources to carry

out “on” / “off” activation. In the first part of this work, we have studied interaction between

NBTXR3 and human colon cancer cells. NBTXR3 penetrates by endocytosis and presents

long residence within endo-lysosomal compartment. Significantly enhance of tumour cell

radiation response is demonstrated after NBTXR3 activation with ionizing radiation.

Compared to controls, irradiated cells having internalized NBTXR3, offer a particular

morphology which is described. In the second part, we present the synthesis of a new hybrid

nanoproduct for photodynamic therapy (PDT), the Pp IX nanocarrier, and the study of its

interaction in vitro on six human cancer cell lines and in vivo on nude mice model

xenografted with three different cancer types. In vitro, Pp IX nanocarrier activated at 630 nm

is more efficient than free Pp IX. Further, this new hybrid nanoproduct enhances

biodistribution of Pp IX, with different kinetic of tumour accumulation between models.

Ultimately, based on these new understanding of interactions between nanoparticles and

cancer cells, both NBTXR3 nanoparticle and Pp IX nanocarrier, offer a breakthrough

approach to create efficient pathways to cancer therapy.

Keywords:

Nanoparticles, Cancer, Nanomedicine, Tumour Cells, Nanoparticles Interaction, Cell

Trafficking, Endosome Residence, Electromagnetic Energy Sources, Therapeutic Window,

Pharmacokinetic, Biodistribution, Radiotherapy, Hafnium Oxide Nanoparticles, Gold

Nanoparticles, Ionizing Radiation, Photodynamic Therapy (PDT), Protoporphyrin IX (Pp IX)

Silica Nanoparticles, Pp IX Nanocarriers, Photosensitizer, Photofrin®, Light Excitation

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NANOMEDECINE EN ONCOLOGIE :

Etude des Interactions Entre les Nanoparticules Activables par des Sources

d’Energie Electromagnétique Externes et les Cellules Cancéreuses pour

Elargir la Fenêtre Thérapeutique

La compréhension des interactions entre les nanomatériaux et les entités biologiques est

fondamentale pour développer des nanoproduits en oncologie. NBTXR3 (nanoparticule inerte

d’oxyde d’hafnium) et protoporphyrine IX (Pp IX) nanotransporteur (nanoparticule de silice

encapsulant le Pp IX, le monomère du Photofrin®) sont des nanoproduits, issus des

plateformes Nanobiotix, développés pour élargir la fenêtre thérapeutique des traitements du

cancer en utilisant des sources d’activation externe de type « on » / « off ». La première partie

de ce travail présente l’étude de l’interaction de NBTXR3 avec des cellules tumorales

coliques humaines. NBTXR3 pénètre par endocytose et persiste dans le compartiment endo-

lysosomial. Une augmentation significative de la réponse cellulaire à la radiothérapie est

démontrée après activation de NBTXR3 par des radiations ionisantes. L’irradiation des

cellules traitées par NBTXR3 entraîne des modifications spécifiques de leur morphologie par

rapport aux contrôles; elles sont décrites ici. Une deuxième partie présente la synthèse d’un

nouvel hybride pour la thérapie photodynamique, le Pp IX nanotransporteur, et étudie son

interaction in vitro avec six lignées de cellules tumorales humaines et in vivo dans un modèle

de souris nude xénogreffée avec trois types tumoraux. In vitro, le Pp IX nanotransporteur

activé à 630 nm est plus efficace que le Pp IX libre. De plus, ce nouvel hybride favorise la

biodistribution du Pp IX, avec une cinétique d’accumulation tumorale différente entre les

modèles. La compréhension des interactions entre nanoparticules et cellules cancéreuses,

apportée par ce travail, contribue à la création de nanothérapies innovantes.

Mots clefs:

Nanoparticules, Cancer, Nanomédecine, Cellules Tumorales, Interaction des Nanoparticules,

Trafic cellulaire, Persistance dans les Endosomes, Sources d’Energie Electromagnétique,

Fenêtre Thérapeutique, Pharmacocinétique, Biodistribution, Radiothérapie, Nanoparticules

d’Oxyde d’Hafnium, Nanoparticules d’Or, Radiation Ionisante, Thérapie Photodynamique,

Nanoparticules de Silice Encapsulant la Protoporphyrine IX (Pp IX), Pp IX Nanotransporteur,

Photosensibilisant, Photofrin®, Excitation par la Lumière

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PhD work was realized in CIFRE convention between:

Nanobiotix, SA

60 rue de Wattignies

75012 PARIS

And

Inserm/Paris VI Units

UMRS 938

Team « Microsatellite Instability and Cancers »

St Antoine Research Center

184 rue du faubourg St Antoine

75012 PARIS

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ABREVIATIONS

3D-CRT: Three-Dimensional Conformal Radiation Therapy 5-ALA: 5-AminoLaevulinic Acid AET: 2-AminoEthaneThiol AFM: Atomic Force Microscopy AIDS: Acquired ImmunoDeficiency Syndrome AUC: Area Under Curve BDSA: 9,10-bis[4’-(4’’-aminostyryl)styryl]anthracene BSA: Bovine Serum Albumin CAM-DR: Cell Adhesion-Mediated Drug Resistance CdS: Cadmium Sulfide CHART: Continuous Hyperfractionated Accelerated Radiation Therapy

Cmax : maximum Concentration CVD: CardioVascular Diseases DDS: Drug-Delivery Systems DEF: Dose Enhancement Factor DHBA: 3,5-DiHydroxyBenzylAlcohol DNA: Desoxy-riboNucleic Acid EBRT: External Beam Radiation Therapy

EC50: 50᧡ Effective Concentration

ECM: ExtraCellular Matrix EGF: Epidermal Growth Factor EGFR: Epidermal Growth Factor Receptor EMDR: Environment-Mediated Drug Resistance EMEA: Europe, Middle East and Africa EPR: Enhanced Permeability and Retention ER: Endoplasmic Reticulum FAP: Familial Adenomatous Polyposis FCS: Fetal Calf Serum FGF: Fibroblast Growth Factor FRET: Fluorescence Resonance Energy Transfer GNP(s): Gold NanoParticle(s) Gy: Gray Hb: Hemoglobin HER: Human Epidermal Receptor HGF: Hepatocyte Growth Factor HIFU: High Intensity Focused Ultrasound HIV: Human Immunodeficiency Virus HNPCC: Hereditary Non-Polyposis Colorectal Cancer

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HP: HaematoPorphyrin HPPH: 2-devinyl-2-(1-Hexyloxyethyl) PyroPheopHorbide HT: Hyperthermia Therapy i.p: intraperitoneal i.v: intravenous ICP-MS: Inductively Coupled Plasma-Mass Spectrometry id/g: injected dose per gram IgG: Immunoglobulin G IGRT: Image Guided Radiation Therapy IL: InterLeukin IMRT: Intensity Modulated Radiation Therapy IR: infra-red Ir: Iridium

LD50: Lethal Dose 50 LDL: Low Density Lipoprotein LITT: Laser-induced Interstitial ThermoTherapy mAb: monoclonal Antibodies MMP: Matrix MetalloProteinases MPS: Mononuclear Phagocyte System MRD: Minimal Residual Disease MRI: Magnetic Resonance Imaging MRT: Mean Resident Time m-THPC: meta-Tetra(HydroxyPhenyl)-Chlorin MTS: 3-(4,5-diMethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-Tetrazolium inner Salt MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide MW: Molecular Weight NCI: National Cancer Institute NE: Nuclear Envelope NGF: Neurotrophin Growth Factor NNI: National Nanotechnology Initiative NO: Nitric Oxide NP(s): NanoParticle(s) NSCLC: Non-Small Cell Lung Cancer OMS: Organisation Mondiale de la Santé ORMOSIL: ORganically MOdified SILicate Pc4: Phthalocyanine 4 PDGF: Platelet-Derived Growth Factor PDT: PhotoDynamic Therapy PEG: PolyEthylene Glycol PG: ProstaGlandin pK: PharmacoKinetic PLGA: Poly(D,L -Lactide-co-Glycolide) Acid QCM: Quartz Crystal Microbalance

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QD: Quantum Dots RES: ReticuloEndothelial System RF: Radio Frequency RNA: RiboNucleic Acid ROS: Reactive oxygen species RTK: Receptor Tyrosine Kinases SBRT: Stereotactic Body Radiation Therapy SCLC: Small Cell Lung Cancer SEM: Scanning Electron Microscopy SFM-DR: Soluble Factor Mediated Drug Resistance SME: Small and Medium Enterprises SPIO: SuperParamagnetic Iron Oxide SWCNT: Single-Walled Carbon NanoTubes t1/2 : half-life TEM: Transmission Electron Microscopy TNF: Tumour Necrosis Factor TPA: Two Photon Absorption US: United States USPIO: Ultra Small SuperParamagnetic Iron Oxide UV: UltraViolet VEGF: Vascular Endothelial Growth Factor WHO: World Health Organization WST-1: (4-[3-(4-Iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) Z: atomic number

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TABLE OF CONTENTS

PART 1: NANOMEDICAL RESEARCH .......................................................................... - 16 -

1. NANOMEDICINE: NANOTECHNOLOGY FOR HEALTH .................................................. - 17 - 1.1. When “nano” meet medicine: the importance of being small .............................. - 17 - 1.2. What is nanomedicine .......................................................................................... - 19 - 1.3. A brief history of nanomedicine ........................................................................... - 20 - 1.4. Focus on nanomaterials in health care ................................................................. - 22 -

1.4.1. Survey of the main diseases around the world .............................................. - 22 - A. Diabetes and cardiovascular disease .............................................................. - 23 - B. Infectious disease ............................................................................................ - 24 - C. Others .............................................................................................................. - 25 -

1.4.2. Where nanotechnology could play a role in health care ............................... - 27 - A. Prevention ....................................................................................................... - 27 - B. Diagnosis ........................................................................................................ - 27 - C. Therapy ........................................................................................................... - 28 - D. Follow-up monitoring ..................................................................................... - 28 -

1.4.3 Which nanomaterials could play a role in health care and why ..................... - 29 - A. Organic-based nanomaterials ......................................................................... - 29 - B. Inorganic-based nanomaterials ....................................................................... - 34 -

1.5. The nanomedicine market .................................................................................... - 37 - 1.6. The future of nanomedicine: health care needs with promising nano-enabled technology impact ....................................................................................................... - 40 -

2. FOCUS ON CANCER DISEASES ........................................................................................ - 42 - 2.1. Cancer ................................................................................................................... - 42 -

2.1.1. Epidemiology ................................................................................................ - 42 - 2.1.2. Four most frequent cancers ........................................................................... - 43 -

A. Lung cancer .................................................................................................... - 43 - B. Colorectal cancer ............................................................................................ - 43 - C. Breast cancer ................................................................................................... - 44 - D. Prostate cancer ................................................................................................ - 45 -

2.1.3. Molecular features of cancer ......................................................................... - 45 - A. Cancer generation ........................................................................................... - 45 - B. Metastasis ....................................................................................................... - 47 -

2.1.4. Anticancer therapy ........................................................................................ - 49 - A. Surgery ........................................................................................................... - 50 - B. Pharmaceuticals .............................................................................................. - 51 - C. Radiotherapy ................................................................................................... - 54 - D. Other biological anticancer approaches.......................................................... - 59 - E. Other physics-based anticancer approaches.................................................... - 60 -

2.1.5. Energy sources for physics-based anticancer approaches ............................. - 61 - A. Ionizing radiation sources .............................................................................. - 61 - B. Laser light source ............................................................................................ - 64 - C. Ultrasound ...................................................................................................... - 65 - D. Magnetic field ................................................................................................. - 66 -

2.2. Narrowness of the therapeutic index: main concern of anticancer treatments ..... - 66 - 2.2.1. Limitations of anticancer treatments ............................................................. - 66 -

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A. Building the therapeutical window ................................................................. - 67 - B. How to reconciliate the tolerated dose and the curative dose ......................... - 71 -

PART 2: ACTIVATED NANOPARTICLES TO TREAT CANCER, A WAY TO ENLARGE THE THERAPEUTIC WINDOW OF ANTICANCER APPROACHES .......................... - 74 -

INTRODUCTION ............................................................................................................... - 75 -

1. NANOBIOTIX COMMITMENT : AN INNOVATION-BASED NANOMATERIAL COMPANY FOR

CANCER TREATMENT ......................................................................................................... - 76 - 2. NANOMATERIAL AND ENERGY INTERACTION: CONCEPT AND RATIONAL FOR DESIGN OF

NANOMATERIAL ................................................................................................................ - 79 - 3. NANOMATERIALS AND BIOLOGICAL MEDIA INTERACTIONS: A NECESSARY UNDERSTANDING

TO BRING THE CONCEPT OF NANOMATERIAL AS “ACTIVE PRODUCT” TO THE CLINIC .......... - 83 - 3.1. The “nano-bio” interface: importance of the protein-corona concept .................. - 83 - 3.2. The nanomaterial delivery at the right time, at the right place and at the right concentration ............................................................................................................... - 85 -

3.2.1. Major obstacles to reach the target: tissues, cells and organelles ................. - 86 - A. Tissues ............................................................................................................ - 86 - B. Cells ................................................................................................................ - 87 - C. Organelles ....................................................................................................... - 88 -

3.2.2. Nanomaterial design for optimized biodistribution and intracellular trafficking: the importance of the nanomaterial size, shape and surface coating....................... - 90 -

A. Nanomaterial size ........................................................................................... - 91 - B. Nanomaterial shape ........................................................................................ - 92 - C. Nanomaterials surface coating: rational to bring a charged, a steric, or a specific targeted coating ................................................................................................... - 92 -

3.3. Toxicological issues: probably the most important concern to address when designing a nanomaterial ............................................................................................. - 99 -

4. SUMMARY OF PHD STUDY: PARTICIPATION TO THE DEVELOPMENT OF TWO TYPES OF

NANOPARTICLES INTENDED TO TREAT CANCER WITH DIFFERENT RATIONAL FOR DESIGN - 102 -

NANOXRAY™ THERAPEUTIC PRODUCTS ALLOW DRAMATIC INCREASE OF X-RAY DOSE ON TUMOUR WITHOUT CHANGING DOSE APPLIED TO HEALTHY TISSUES. NANOXRAY™ INCREASES LOCAL DESTRUCTION OF THE TUMOUR WITHOUT MODIFYING HEALTHY TISSUES ............................................................ - 107 -

1. NANOPARTICLES ACTIVATED BY X-RAY: NBTXR3 MECHANISM OF ACTION .............. - 108 - 2. METALLIC NANOPARTICLES ACTIVATED BY IONIZING RADIATION TO TREAT CANCER . - 111 -

Gold nanoparticles literature survey.......................................................................... - 111 - 3. ENHANCEMENT OF RADIATION ACTIVITY OF NANOXRAY

TM ........................................ - 120 - 3.1. “Endosomes Bioavailability as Key Parameter for Optimal Efficacy of Radiation Enhancer Nanoparticles on Human Colon Cancer Cells” ......................................... - 120 - Virginie Simon, Ping Zhang, Laurence Maggiorella, Agnès Pottier, Elsa Borghi, Laurent Levy, Julie Marill ; In Preparation ............................................................................ - 120 - 3.2. Discussion .......................................................................................................... - 123 -

4. CONCLUSION ............................................................................................................... - 125 -

PP IX SILICA NANOPARTICLES CREATE DIFFERENTIAL BIODISTRIBUTION BETWEEN TUMOUR AND HEALTHY TISSUES IN TERMS OF TUMOUR UPTAKE AND SUBCELLULAR LOCALIZATION: PHOTODYNAMIC THERAPY THERAPEUTIC WINDOW ENLARGEMENT .............................................................. - 127 -

1. PHOTODYNAMIC THERAPY MECHANISM OF ACTION ..................................................... - 128 -

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2. PHOTOSENSITIZER: LIGHT EXCITATION ........................................................................ - 129 - 2.1. Optimum for superficial tumours: light penetration in tissues ........................... - 129 - 2.2. Chemistry improvement: second, third generation of photosensitizer ............... - 131 - 2.3. Presentation of approved photosensitizers and current research ........................ - 133 -

2.3.1. Porphyrin, texaphyrin and chlorin photosensitizers in clinics .................... - 133 - 2.3.2. Others trends ............................................................................................... - 135 -

3. SYSTEMIC ADMINISTRATION OF PHOTOSENSITIZER FOR VERY LOCALIZED TREATMENT . - 137

- 3.1. Half life of generated ROS and distance for ROS effect: subcellular localization of the photosensitizer is required ................................................................................... - 137 - 3.2. Pharmacokinetics, pharmacodynamic and subsequent adverse effects of photosensitizer: example of Photofrin® ..................................................................... - 139 -

3.2.1 Pharmacokinetic ........................................................................................... - 139 - A. Absorption-bioavailability ........................................................................ - 140 - B. Metabolism ............................................................................................... - 140 - C. Excretion ................................................................................................... - 140 - D. Others ....................................................................................................... - 141 -

3.2.2. Pharmacodynamic ....................................................................................... - 141 - A. Distribution in in vivo models .................................................................. - 141 -

3.2.3. Side effects .................................................................................................. - 142 - 4. NANOCARRIERS PLAYERS: A WAY TO ENHANCE SELECTIVITY AND SPECIFICITY OF

PHOTOSENSITIZER TO IMPROVE THEIR TUMOUR BIOAVAILABILITY .................................. - 143 - 4.1. Silica-based nanocarriers of photosensitizers: literature survey ........................ - 143 - 4.2. Selection the of simplest process route to design Pp IX silica-based nanocarrier - 146 - “One Pot Synthesis of New Hybrid Versatile Nanocarrier Exhibiting Efficient Stability in Biological Environment for Use in Photodynamic Therapy” ............................... - 146 - Edouard Thiénot, Matthieu Germain, Kelthoum Piejos, Virginie Simon, Audrey Darmon, Julie Marill, Elsa Borghi, Laurent Levy, Jean-François Hochepied, Agnès Pottier ; Submitted .................................................................................................................. - 146 - 4.3. Discussion .......................................................................................................... - 148 -

5. INTERACTION OF PP IX SILICA NANOPARTICLES WITH BIOLOGICAL SYSTEMS .............. - 150 - 5.1. “Pp IX Silica Nanoparticles Demonstrate Differential Interactions with In Vitro Tumour cell Lines and In Vivo Mouse Models of Human Cancers” ........................ - 150 - Virginie Simon, Corinne Devaux, Audrey Darmon, Thibault Donnet, Edouard Thiénot, Matthieu Germain, Jérôme Honnorat, Alex Duval, Agnès Pottier, Elsa Borghi, Laurent Levy, Julie Marill ; Photochemistry, Photobiology, 2009 ......................................... - 150 - 5.2. Discussion .......................................................................................................... - 153 -

6. GENERALS PDT CONCLUSIONS AND PERSPECTIVES ..................................................... - 155 -

GENERALS CONCLUSIONS AND PERSPECTIVES .................................................. - 157 -

BIBLIOGRAPHY ............................................................................................................. - 161 -

RESUME DETAILLE DE LA THESE ............................................................................ - 172 -

- 16 -

PART 1: NANOMEDICAL RESEARCH

- 17 -

1. NANOMEDICINE: nanotechnology for health

1.1. When “nano” meet medicine: the importance of being small

A key date for nanotechnology and more specifically for nanotechnology applied to

medicine is the year 1959, where R. Feynman in its historic lecture, ‘There’s Plenty of Room

at the Bottom, An Invitation to Enter a New Field of Physics”, given at the California Institute

of Technology, pointed out the promises and potentials of nanotechnology: “When we get to

the very, very small world---say circuits of seven atoms---we have a lot of new things that

would happen that represent completely new opportunities for design. Atoms on a small scale

behave like nothing on a large scale, for they satisfy the laws of quantum mechanics. So, as

we go down and fiddle around with the atoms down there, we are working with different laws,

and we can expect to do different things. We can manufacture in different ways. We can use,

not just circuits, but some system involving the quantized energy levels, or the interactions of

quantized spins, ...”

His discussion may be considered as the earliest vision of nanotechnology applied to

medicine as he raised the question and encouraged the vision of “manufacture of an object

that can manoeuvre at the level of biological cells” (Webster et al., 2007).

Since, nanotechnologies have created a “nanoworld” and are today able to generate

systems, from the very simple to a more complex and elaborated one, as illustrated by the

emergence of supramolecular chemistry which is based on molecular recognition and self-

assembly processes (Lehn Jean-Marie, 1995). Nanomaterials, due to their very specific small

size are able to interact with excessively small systems; and biological systems are today the

playground for nanomaterials applications having for ambition to address unmet needs in

biology. When materials size matches the size of biological entities, pathways between the

two systems may be established, the nanomaterials being able to gain access and even to

operate within cells. Indeed, controlling and manipulating things at the nanometer scale

allows exploring and interacting at the cellular level with unprecedented fashion.

When looking at the nanometer scale, one speaks of objects on the order of one billion

of a meter. To put this size in perspective, a human hair is approximately 80.000 nm wide,

and a red blood cell is approximately 7.000 nm wide. Atoms are smaller than 1 nm, whereas

many molecules including some proteins range between 1 nm and larger (Sahoo et al., 2007).

Generally, the sizes of nanomaterials are comparable to those of viruses, desoxy-ribonucleic

- 18 -

acid (DNA), and proteins, while microparticles are comparable to cells, organelles, and larger

physiological structures (Buzea et al., 2007). Figure 1 and Figure 2 give an illustration of the

structures down to the nanometer scale.

Figure 1: Relative sizes of small objects including (a) cockroach, (b) human hair, (c) pollen grain, (d) red blood cells, (e) aggregates of half shells of palladium, (f) superlattice of cobalt nanocrystals, and

(g) aspirin molecule (Emerich et al., 2006)

Figure 2: Schematics illustrating a microparticle of 60 µm diameter, about the size of a human hair (shown in the left at scale) and the number of NPs with diameter of 600 nm and 60 nm having the

same mass as microparticle of 60 µm diameter (Buzea et al., 2007)

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- 19 -

The strength of nanomaterial in medicine lies in its ability to operate on the same

small scale as all the intimate biochemical functions involved in the growth, development and

ageing of the human body (Figure 3). It is expected to provide a new framework for

diagnosing, treating and preventing disease.

Figure 3: Sub-cellular nanoparticles size

Nanomaterials in medicine, are function-oriented: they may bring an enabling function

– drug delivery systems for instance, aiming to improve the benefice over risk ratio

comparatively to free drugs – or they may bring new modalities, due to their specific

properties arising at the nanometer scale, which are expected to address breakthrough

potential for patient care.

Anyhow, those nanomaterials originated from the technological world have to enter

the biological world if they have to realize all their promises. Indeed, design of nanomaterials

matching the size of most biological entities, is the foundation for creating pathways and

effective interactions between the two systems. But lot needs to be done to establish proper

recognition and create effective cross-talk between engineered nanomaterials and biological

entities for the safe and efficient use of “nano” in medicine.

1.2. What is nanomedicine

The term nanomedicine can be traced back to the late 1990s; according to the Science

Citation Index (Institute for Scientific Information, Thompson, Philadelphia, PA, United

States; US) the first research publications that use this term appeared in the year 2000. With

- 20 -

research programs, conferences and journals focusing on nanomedicine for a number of years

now, it has become clear that nanomedicine is more than a semantic fashion, though it was

difficult to find a precise definition for this field with its blurred borderlines encompassing

biotech and microsystems technology.

In general, two concepts can be distinguished. Some experts define nanomedicine very

broadly as a technology that uses molecular tools and knowledge of the human body for

medical diagnosis and treatment. Others prefer an emphasis on the original meaning of

nanotechnology as one that makes use of physical effects occurring in nanoscale objects that

exist at the interface between the molecular and macroscopic world in which quantum

mechanics still reigns. This second concept is commonly adopted and nanomedicine may be

defined as the use of nanoscale or nanostructured materials in medicine that according to their

structure have unique medical effects, for example, the ability to cross biological barriers or

the passive targeting of tissues (Wagner et al., 2006).

Such medical effects are not strictly limited to a size range below 100 nanometers

(nm). Therefore, unlike the physical definition of nanotechnology, which is restricted to

objects with dimensions in the range of 1 nm to 100 nm, structures and objects up to 1.000 nm

in size are included. Such a definition also seems to be justified from a technical point of view

because the control of materials in this size range not only results in new medical effects but

also requires novel, scientifically demanding chemistry and manufacturing techniques.

1.3. A brief history of nanomedicine

Important events have contributed to the emergence of nanomedicine and to its

expansion to the point we stand today. Some of them are highlighted in the followings.

In 1986, the Atomic Force Microscopy (AFM) was invented which had subsequently

allowed for unprecedented control over nanomaterials design and characterization.

In 1987, the first university symposium “Exploring Nanotechnology” was opened at

the Massachusetts Institute of Technology, Cambridge, MA, US.

In 1988, the first university course, “Nanotechnology and Exploratory Engineering”,

was created at Stanford University, Palo Alto, CA, US.

In 1990, the first journal called Nanotechnology was edited.

- 21 -

In 1996, the first “nano-bio” conference by the International Business

Communications “Biological Approaches and Novel Applications for Molecular

Nanotechnology” hold in San Diego, CA, US.

In 2000, US President Clinton announced the creation of the US National

Nanotechnology Initiative (NNI). NNI is a multi-agency umbrella programs to build, to

characterize, and to understand nanoscale devices. The NNI lists medicine, manufacturing,

material sciences, information technology, energy, and environmental sciences as target

beneficiaries. The program slated to spend nearly $1 billion in fiscal year 2005 as compared

with $464 million in 2001 (Sahoo et al., 2007).

In 2003, Robert Freitas Jr., who was one of the earliest contemporary proponents of

nanoscience, in his book Nanomedicine, envisaged the future of nanoscience as it applies to

medicine in “three overlapping and progressively more powerful technologies; the first, in

the immediate future, refers to addressing medical problems by using nanostructures:

materials that can be manufactured today; then, in the near future, he anticipated advances in

molecular medicine and botics; and, finally, in the long term, molecular machine systems and

nanorobots joining the medical armamentarium” (Asiyanbola et al., 2008).

In 2003, Pr. Paras N. Prasad, the executive director of the Institute for Lasers,

Photonics and Biophotonics (State University of New York) and one of the world’s leading

authorities on nanotechnology, in his book Introduction to biophotonics covered current and

future applications based on light-activated therapies. Since, Biophotonics offer exciting

opportunities for fundamental research to probe intercellular interactions as well as to produce

novel biotechnology to advance human health, particularly in the new field of nanomedicine.

In 2006, the first international journal in nanomedicine, International Journal of

Nanomedicine, was edited.

In 2008, Nanobiotix company, a spin-off of the State University of New York

(SUNY) at Buffalo that has been incorporated in 2003, announced that the European service

of patents have delivered their patent for new nanoXrayTM nanoparticles activated by X-ray

for cancer care. NanoXrayTM offers a dramatic innovation in cancer therapy, based on a

technology that is designed to allow destruction of cancer cells by inert particles.

In 2009, Mauro Ferrari , a specialist in the field of nanomedicine, gave an interesting

vision when answering the following question: How do you think the nanomedicine field will

influence the treatment of disease? “I think medicine is really going to change in five to ten

years, with much greater emphasis on early detection so that we can catch diseases before

they develop to the point where it’s very hard and expensive and many times impossible to

- 22 -

control. And that will be enabled by nanotechnology. We will be able to develop personalized

approaches for cancer and for other diseases, and, again, nanotechnology will be a necessary

lever of all of that. Everything is changing, and luckily for the better” (Ferrari et al., 2009).

Nowadays, nanomedicine is perceived as embracing five main sub-disciplines that in

many ways are overlapping and underpinned by the following common technical issues:

analytical tools; nanoparticles-imaging; nanomaterials and nanodevices; novel therapeutics

and drug delivery systems; regulatory, clinical and toxicological issues.

1.4. Focus on nanomaterials in health care

1.4.1. Survey of the main diseases around the world

Figure 4 shows main causes of human death around the world and according to

continental area. Among them, main diseases leading to human deaths are highlighted. In

Sub-Saharan Africa, infectious and parasitic diseases, including measles and malaria, are

more frequent causes of death than elsewhere. Respiratory infection disproportionately affects

people living in Southeast Asia and Sub-Saharan African. These two regions are also

particularly hit by maternal conditions and perinatal conditions. The Asia and the West

Pacific region have a rate of non-communicable respiratory diseases, such as chronic

bronchitis and emphysema, which is nearly 2.5 times higher than the rest of the world.

Western Europe has a greater proportion of deaths due to heart (cardiovascular) disease and

cancer (malignant and other neoplasms).

Figure 4 : What people die? (UC Atlas of Global Inequality)

- 23 -

In developing nations, inferior sanitary conditions and lack of access to modern

medical technology make death from infectious diseases more common than in developed

countries.

In the world, over 30.000 children under the age of five die each day from preventable

causes related to conditions of extreme poverty. Malnutrition is estimated to contribute to

more than one third of all child deaths, although it is rarely listed as the direct cause. Infection

– particularly frequent or persistent diarrhoea, pneumonia, measles and malaria – also

undermines a child's nutritional status.

The main causes of mortality in the world for adults aged between 15 and 60 are

acquired immunodeficiency syndrome (AIDS), cardiovascular disease and tuberculosis. For

people aged more than 60 the highest causes of mortality are cardiovascular diseases, cancer

and diabetes (Figure 5 and Figure 6).

The main diseases are briefly summarized in the following; cancer will be reviewed in

a specific chapter.

Figure 5 : Leading causes of mortality among adults into the world in 2002 (OMS, 2009)

A. Diabetes and cardiovascular disease

Diabetes

Diabetes is a chronic disease which occurs when the pancreas does not produce

enough insulin, or when the body cannot effectively use the insulin it produces. In 2000, at

least 171 million people worldwide had diabetes. This figure is likely to more than double by

2030 (366 million estimated). In developing countries the number of people with diabetes will

increase by 150 % in the next 25 years. The global increase in diabetes will occur because of

Rank Cause Deaths (000)

2279

1332

1036

814

783

672

473

382

352

343

HIV/AIDS

Ischaemic heart disease

Tuberculosis

Road traffic injuries

Cerebrovascular disease

Self-inflicted injuries

Violence

Cirrhosis of the liver

Lower respiratory infections

Chronic obstructive pulmonary disease

1

2

345

6

7

8

9

10

Mortality-Adults Aged 15-59


2279

1332

1036

814

783

672

473

382

352

343

HIV/AIDS


Tuberculosis




Violence




1

2

345

6

7

8

9

10



5825

4689

2399

1396

928

754

735

605

495

477





Trachea, bronchus, lung cancers

Diabetes mellitus

Hypertensive heart disease

Stomach cancer

Tuberculosis

Colon and rectum cancers

1

2

345

6

7

8

9

10

Mortality-Adults Aged 60+


5825

4689

2399

1396

928

754

735

605

495

477






Diabetes mellitus


Stomach cancer

Tuberculosis


1

2

345

6

7

8

9

10



2279

1332

1036

814

783

672

473

382

352

343

HIV/AIDS


Tuberculosis




Violence




1

2

345

6

7

8

9

10



2279

1332

1036

814

783

672

473

382

352

343

HIV/AIDS


Tuberculosis




Violence




1

2

345

6

7

8

9

10



5825

4689

2399

1396

928

754

735

605

495

477






Diabetes mellitus


Stomach cancer

Tuberculosis


1

2

345

6

7

8

9

10



5825

4689

2399

1396

928

754

735

605

495

477






Diabetes mellitus


Stomach cancer

Tuberculosis


1

2

345

6

7

8

9

10


- 24 -

population ageing and growth, and because of increasing trends towards obesity, unhealthy

diets and sedentary lifestyles. In developed countries most people with diabetes are above the

age of retirement, whereas in developing countries those most frequently affected are aged

between 35 and 64 (WHO world health report, 2009).

Cardiovascular diseases (CVD)

CVD are a group of disorders of the heart and blood vessels and include coronary

heart, cerebrovascular, peripherical arterial, rheumatic heart, congenital heart disease and

deep vein thrombosis and pulmonary embolism. CVD are the number one cause of death

globally: more people die annually from CVD than from any other cause. An estimated 17.5

million people died from CVD in 2005, representing 30% of all global deaths. Of these

deaths, an estimated 7.6 million were due to coronary heart disease and 5.7 million were due

to stroke. Over 80% of CVD deaths take place in low and middle income countries and occur

almost equally in men and women. By 2015, almost 20 million people will die from CVD,

mainly from heart disease and stroke. CVD are projected to remain the single leading causes

of death (WHO world health report, 2009).

B. Infectious disease

AIDS

Acquired immune deficiency syndrome or acquired immunodeficiency syndrome

(AIDS) is a disease of the human immune system caused by the human immunodeficiency

virus (HIV). 33 million people were living with HIV in 2007 all around the world with 2

million of deaths and 2.7 million newly infected (UNAIDS, 2009).

Malaria

Malaria is caused by a parasite called Plasmodium, which is transmitted via the bite of

infected mosquitoes. In the human body, the parasites multiply in the liver, and then infect red

blood cells. There were 247 million cases of malaria in 2006, causing nearly one million

deaths, mostly among African children. Approximately half of the world's population is at

risk of malaria, particularly those living in lower-income countries (WHO world health report,

2009).

- 25 -

Measles

Measles is a leading cause of death among young children even though a safe and

cost-effective vaccine is available to prevent the disease. In 2007, there were 197 000 measles

deaths globally. More than 95% of measles deaths occur in low income countries with weak

health infrastructures. Measles vaccination efforts have reaped major public health gains,

resulting in a 74% drop in measles deaths between 2000 and 2007 worldwide - with a drop of

about 90% in the eastern Mediterranean and Africa regions (WHO world health report, 2009).

C. Others

In developed countries with an ageing population, there is rising prevalence in

diseases of the central nervous system. Parkinson’s disease, senile dementia or Alzheimer’s

disease, arthritis and ocular diseases are also important mortality causes.

- 26 -

Figure 6: Health map world in 2008

http://www.dni.gov/nic/PDF_GIF_otherprod/ICA_Global_Health_2008_foldout.pdf

- 27 -

1.4.2. Where nanotechnology could play a role in health care

A. Prevention

In medicine, prevention is the action taken to decrease the chance of getting a disease

or condition. For example, cancer prevention includes avoiding risk factors (such as smoking,

obesity, lack of exercise, and radiation exposure) and increasing protective factors (such as

getting regular physical activity, staying at a healthy weight, and having a healthy diet) (NCI).

New diagnostic tests making use of nanotechnology to quantify disease-related

biomarkers could offer an earlier and more personalised risk assessment before symptoms

show up. Supported by such an analysis and bioinformatics, health professionals could advise

patients with an increased risk to take up a personalised prevention program. People with an

increased risk for a certain disease could benefit from regular personalised check-ups to

monitor changes in the pattern of their biomarkers. Nanotechnology could improve in vitro

diagnostic tests by providing more sensitive detection technologies or by providing better

nano-labels that can be detected with high sensitivity once they bind to disease-specific

molecules present in the sample. Diseases with no secretion of biomarkers into blood or urine

will require imaging procedures of high specificity for their early detection. One well-known

example used already is X-ray mammography for the early detection of breast cancer. Novel

targeted imaging agents, precisely homing in on diseased cells, promise a much higher

sensitivity than today's imaging procedures, making possible the detecting of disease at an

even earlier stage.

B. Diagnosis

Diagnosis is the process of identifying a disease, such as cancer, from its signs and

symptoms (NCI).

If a medical check-up had found an indication or a hint of symptoms for a disease, it is

important that “false positives” are excluded by applying more specific diagnostic procedures.

In this case, molecular imaging, which makes use of specific targeted agents, plays a crucial

role for localisation and staging of a disease, or – equally important – for ascertaining the

health of a patient. Here, nanotechnology could help to design a plethora of very specific

imaging agents over the next ten years. Conceptually novel methods, combining biochemical

techniques with advanced imaging and spectroscopy provide insight to the behaviour of single

diseased cells and their microenvironment for the individual patient. This could lead to

- 28 -

personalised treatment and medication tailored to the specific needs of a patient. The main

advantage of nanomedicine on quality of life and on costs for healthcare is earlier detection of

a disease, leading to less severe and costly therapeutic demands, and an improved clinical

result. However, once a disease is diagnosed, therapeutic action is required. A decision needs

to be taken as to which cure offers the best therapeutic ratio (risk/benefit) for the patient.

Here, diagnostic imaging procedures provide crucial input for clinical decision taking and

therapy planning.

C. Therapy

In many cases, therapy will not be restricted to medication only but requires more

severe therapeutic action such as surgery or radiation treatment. Planning of therapeutic

interventions will be based on imaging, or may be performed under image guidance. Here,

nanotechnology will lead to a miniaturisation of devices that enable minimally invasive

procedures and new ways of treatment. The possibilities range from minimally invasive

catheter-based interventions to implantable devices. Targeted delivery systems and

nanotechnology-assisted regenerative medicine will play the central role in future therapy.

Targeted delivery agents will, as example, allow a localised therapy which targets only the

diseased cells, thereby increasing efficacy while reducing unwanted side effects. Thanks to

nanotechnology, pluripotent stem cells and bioactive signalling factors will be essential

components of smart, multi-functional implants which can react to the surrounding micro-

environment and facilitate site-specific, endogenous tissue regeneration (making lifelong

immune-suppressing medication obsolete). Imaging and biochemical assay techniques will be

used to monitor drug release or to follow the therapy progress. This therapeutic logic will lead

to the development of novel, disease modifying treatments that will not only significantly

increase quality of life but also dramatically reduce societal and economic costs related to the

management of permanent disabilities.

D. Follow-up monitoring

Medical reasons may call for an ongoing monitoring of the patient after completing

the acute therapy. This might be a regular check for reoccurrence, or, in the case of chronic

diseases, a frequent assessment of the actual disease status and medication planning.

Continuous medication could be made more convenient by implants, which release drugs in a

controlled way over an extended length of time. In vitro diagnostic techniques and molecular

- 29 -

imaging play an important role in this part of the care-process, as well. Biomarkers could be

systematically monitored to pick up early signs of reoccurrence, complemented by molecular

imaging where necessary. Oncology is one of the areas where these techniques are already

being evaluated today. Some types of tumours can be controlled by continuous medication

extending life expectancy. However, in the case of drug resistance, signs of disease

progression can be immediately picked up and alternative treatments can be prescribed.

1.4.3 Which nanomaterials could play a role in health care and why

Due to the advance in nanotechnology, the preparation of a large variety of

nanomaterials is today achievable. Those nanomaterials offer various unique properties that

are able to meet important medical needs.

Nanomaterials may be classified in two main groups, depending on the core of the

material: the inorganic and the organic nanomaterials.

Their way of production may be rationalized within two mains approaches. The Top-

Down approach involves creating smaller materials by using larger ones by for example

moulding or etching. Nanolithography is a typical example of the top-down approach. The

Bottom-Up approach involves arranging small components such as atoms or molecules in

more complex structures. Molecular self-assembly is a typical example of the bottom-up

approach.

A. Organic-based nanomaterials

Organic-based nanomaterials encompass a large variety of structures having as

common feature the ability to carry and eventually deliver molecules in a controlled way. The

most advanced nanomaterials for clinical applications are the liposomes, the micelles, the

polymeric nanoparticles and the dendrimers. A brief overview of each type of structure is

proposed in the following.

- 30 -

LIPOSOMES

Figure 7 : Liposomes structures with phospholipid bilayer enclosing a hydrophilic core. A- Schematic diagram of lipids incorporated into a bilayer membrane to form a liposome (Huang et al., 2008 and

Husseini et al., 2008). B- Cryo-TEM images of liposomes (Li et al., 1998)

Liposomes are vesicles which consist of one to several, chemically-active lipid bilayers. Drug

molecules can be encapsulated and/or solubilised within the bilayers according to their

hydrophilic/lipophilic balance. Drugs incorporated within a liposome are effectively protected

from premature degradation. Drug delivery may be triggered by different stimuli (temperature,

pressure for instance) according to the composition of the lipid bilayer. “Channel” proteins can

also be incorporated in liposome membrane to act as size-selective filters allowing the diffusion

of small solutes such as ions, nutrients and antibiotics. The drug molecule, however, is able to

diffuse through the channel, driven by the concentration difference between the interior and the

exterior of the liposome.

Main applications:

Nanocarriers

Drug Delivery Systems

- 31 -

MICELLES

Figure 8 : A- Micelles structures with phospholipids monolayer enclosing a hydrophobic core (Husseini et al., 2008) B- CryoTEM images of mixed micellar structures

Micelles are vesicles formed from aggregation of surfactant molecules. In aqueous solution

(oil-in-water micelles), the hydrophilic “head” of surfactant molecules are in contact with the

surrounding solvent, sequestering the hydrophobic tail region in the micelle core. Drugs,

particularly hydrophobic drugs, can be trapped in the core of a micelle and transported at

concentrations even greater than their intrinsic water solubility. A further feature which makes

micelles attractive is that their size and shape can be changed by, for example, addition of co-

surfactant, temperature, surfactant concentration. Chemical techniques using cross-linking

molecules can improve the stability of the micelles and their temporal control.

Main applications:

Nanocarriers


50 nm

- 32 -

POLYMERIC NANOPARTICLES

Figure 9 : Scanning electron microscopy (SEM) image of poly(D,L -lactide-co-glycolide) (PLGA) spheres (Zhao et al., 2007)

Polymeric nanoparticles encompass a large variety of structures such as nanogels, albumin,

polymer, and the lipid-like systems, that all can serve in the transport process. The polymeric

nanoparticles typically function to enclose or encapsulate a drug with a controlled release time.

Ideally, polymeric nanoparticles should be biodegradable, like those made of polylactic acid,

polyalkylcyanoacrylate, polyglycolic acid, or polyethylene oxide, among others (Brewer et al.,

2007).

Main applications:

Nanocarriers


- 33 -

DENDRIMERS

Figure 10 : A-Dendrimers structures (Sanvicens et al., 2008) B- Photograph of 3,5-dihydroxybenzylalcohol (DHBA) based dendrimers on silicon substrate. Scale: 5×5 mm (Vassilieff et

al., 2006)

Dendrimers are a major architectural class of nanoscale chemical polymers. The term

dendrimer describes a large, synthetically produced precisely defined polymer in which the

atoms are arranged in many branches and sub-branches radiating out from a central core.

Dendrimers are built from a starting atom, for example nitrogen, to which carbon and other

elements are added by a repeating series of chemical reactions that produce a spherical

branching structure. The core chemistry determines the solubilizing properties of the cavity

within the core, whereas external chemical groups determine the solubility and chemical

behaviour of the dendrimer itself.

Main applications:

Nanocarriers


- 34 -

B. Inorganic-based nanomaterials

Inorganic-based nanomaterials are of particular relevance for medical applications.

Indeed, exclusive properties arise from the core of the materials specifically at the nanometer

scale due to the so called quantum-size effect. As the surface-area-to-volume ratio increases

when reducing the size of the materials, new electronic or magnetic properties may be

observed and exploited for specific medical applications. Examples of the most developed

inorganic nanoparticles for nanomedicine are reported below.

SUPERPARAMAGNETIC NANOPARTICLES

Figure 11 : Transmission electron microscopy (TEM) image of superparamagnetic ion oxide

(SPIO) Nanoparticles

Small iron oxide nanoparticles (SPIO) or ultra-small iron oxide nanoparticles

(USPIO) are the most exploited superparamagnetic oxide nanoparticles in medicine.

They adopt the magnetite (Fe3O4) or maghemite (Fe2O3) spinelle structure. When

particles size goes down 10 nm, superparamagnetic property occurs. Such property is

of particular relevance for imaging using standard magnetic resonance imaging (MRI)

technology due to changes in the spin-spin relaxations of neighbouring water

molecules. Such nanoparticles are also developed for activation by high frequency

alternative magnetic field for hyperthermia therapy.

Main applications:

Contrast Agents,

Therapeutic Agents,

Nanocarriers

- 35 -

QUANTUM DOTS

Figure 12 : Quantum dots (QD) optical properties (Sanvicens et al., 2008)

Quantum dots (QD) are colloidal fluorescent semiconductor nanocrystals with size ranging

from about 2 nm up to 10 nm. The central core of QD consists of combination of elements

from groups II–VI (CdSe, CdTe, CdS, PbSe, ZnS and ZnSe) or III–V (GaAs, GaN, InP and

InAs) of the periodic table. The nanoparticle core is generally “overcoated” with a layer of

ZnS. QD show size and composition-tuneable emission spectra and high quantum yield. They

are resistant to photobleaching and show exceptional resistance to photo and chemical

degradation. All these characteristics make QD excellent contrast agents for imaging and labels

for bioassays.

Main applications:

Contrast Agents,

Nanocarriers

Labels for Bioassays

- 36 -

METALLIC NANOPARTICLES

A B

50 nm

A B

50 nm

Figure 13 : A-Gold nanoparticles. B-TEM Images

Both gold and silver nanoparticles have received renewed interest because of their fascinating

localized surface plasmon resonance properties, which can generate a strong electromagnetic

field in the vicinity of a particle surface on irradiation with light. This light-induced field can

enhance the intensity of Raman scattering by up to a billion times, enabling the development of

optical probes for detecting biomarkers indicative of specific diseases at low level.

These nanoparticles have been further developed into colorimetric sensors, contrast agents for

imaging modalities based on optical coherence, photoacoustics, two-photon fluorescence; and

photothermal agents for cancer treatment and controlled release of drugs.

Main applications:

Contrast Agents,

Therapeutic Agents

Nanocarriers

Labels for Bioassays

- 37 -

1.5. The nanomedicine market

A characterizing feature of nanotechnology today, is its enabling function to add new

functionality to existing products, making them more competitive. As measuring the added

value of nanotechnology to a product is not possible, it has become common praxis in

nanotechnology business studies to take the total sales of nanotechnology-enhanced products

as a measure of the economic importance of nanotechnology in an industrial sector.

Data published in 2006 (Wagner et al.) proved that commercialization efforts were

already significant, with more than 150 start-ups and small and medium enterprises (SME)

pursuing focused nanomedicine research and development projects and 38 nanotechnology-

enabled products currently on the market were reported with total sales valued at $6.8 billion.

Taking into account the pipeline of nanomedicine products that were in an advanced

development stage, it was predicted that the market will grow to around $12 billion in the year

2012. Figure 14 summarizes some of nanomedicine products on the market (Wagner et al.,

2006) and Figure 15 reports a list of product entering preclinical or clinical phase (Zhang et

al., 2007).

Currently, one can see that nanomedicine is dominated by drug delivery systems using

organic-based nanomaterials as nanocarriers. Inorganic-based nanomaterials are mostly

developed for other health care applications such as in vitro diagnostic and in vivo imaging. In

the field of in vitro diagnostic, the most widely used nanotechnology product is colloidal gold

in lateral flow assays, which is used in rapid tests for pregnancy, ovulation, HIV and other

indications. Further magnetic nanoparticles are used for cell sorting applications in clinical

diagnostics. Nanotechnology-based contrast agents, albeit often cited as important examples

for nanomedicine, are a niche market and all of the marketed contrast agents consist of SPIO

nanoparticles for MRI.

Interestingly, nanomaterials are used to develop novel therapies or drugs in which the

nanomaterial plays the pivotal therapeutic role. Here, the specific property offered by the

materials at the nanometer scale is fundamental. A prominent example for such a

nanomedicine is nanoparticles-based magnetic hyperthermia being developed for the

treatment of cancer by the startup Magforce (Berlin). In this treatment, aminosilane-coated

magnetic nanoparticles are injected into the tumour and subsequently heated with a newly

developed magnetic field applicator. Nanoparticles coated with aminosilane are taken up

faster by tumour cells than by normal cells. The tumour cells are destroyed by the heat

generated by the nanoparticles upon activation.

- 38 -

Figure 14 : Nanomedicine products on the market (Wagner et al., 2006)

In vitro diagnostics

Lateral flow tests

Clinical cell separation

Schering, DE

Advanced Magnetics, US and Guerbet, FR

Advanced Magnetics, Guerbet

Liver cancer

Liver cancer

Imaging of abdominal structures

Iron NP

Iron NP

Iron NP

Resovist

Feridex/Endorem

Gastromark/Lumirem

Dentspley, GB

3M Espe, DE

Heraeus Kulzer, DE

Sybron Dental Specialties, US

Ivoclar Vivadent, LI

Osartis, DE

Aap Implantate, DE

Orthovita, US

Nucryst, US

Dental filling material


Dental restoration

Dental repair

Dental repair

Bone defects

Bone defects

Bone defects

Antimicrobial wound care

NP composite

NP composite

NP-containing dental prothesis

NP composite

NP composite

NP-hydroxyapatite

NP-hydroxyapatite

NP-hydroxyapatite

Silver NP

Ceram X duo

Filtek Supreme

Mondial

Premise

Tetric EvoCeram

Ostim

Perossal

Vitoss

Acticoat

Biomaterials

Enzon, US

InterMune, US

Gilead, US and Fujisawa, JP

Gilead

Ortho Biotech, US and Schering-Plough, US

SkyePharma, GB and Enzon

Berna Biotech, CH

Berna Biotech

Zeneus Pharma, GB

QLT, CA and Novartis, CH

Novavax, US

Enzon

Amgen, US

Enzon

Nektar, US and Hoffmann-La Roche, CH

Enzon, Schering-Plough

OSI Pharmaceuticals and Pfizer, US

Nektar, Pfizer

TEVA Pharmaceuticals, IL

Genzyme, US

Elan Drug Delivery and Merck, US

Elan Drug Delivery, WyethPharmaceuticals, US

Elan Drug Delivery, Abbott, US

SkyePharma, First Horizon Pharmaceuticals, US

Abraxis Bioscience, US and AstraZeneca, GB

Fungal infections

Fungal infections

Fungal infections

Kaposi sarcoma

Cancer, Kaposi sarcoma

Cancer

Hepatitis A

Influenza

Breast cancer

Macular degeneration

Mesopausal therapy

Immunodeficiency disease

Febrile neutropenia

Leukemia

Hepatitis C

Hepatitis C


Acromegaly

Multiple sclerosis

Chronic kidney disease

Antiemetic

Eating disorders

Immunosuppressant

Lipid regulation

Lipid regulation

cancer

Amphotericin B/ lipid complex

Amphotericin B/ lipid colloidal

Liposomal amphotericin B

Liposomal daunorubicin

Liposomal doxorubicin

Liposomal cytarabine

Virosomal hapatitis vaccine

Virosomal influenza vaccine


Liposomal verteporfin

Estradiol in micellar NP

PEG-adenosine deaminase

PEG-G-CSF

PEG-asparaginase

PEG-α-interferon 2a

PEG-α-interferon 2b

Pegylated anti-VEGF aptamer

PEG-HGH

Copplymer of alanine, lysine, glutaminicacid and tyrosine

Crosslinked polyresin

NP aprepitant

NP megesterol acetate

NP megesterol sirolimus

NP fenofibrate

NP fenofibrate

Paclitaxel protein bound NP

Abelcet

Amphotec

Ambisome

DaunoXome

Doxil

Depocyt

Epaxal Berna

Inflexal V Berna

Myocet

Visudyne

Estrasorb

Adagen

Neulasta

Oncaspar

Pegasys

PEG-Intron

Macugen

Somavert

Copaxone

Renagel

Emend

MegaceES

Rapamune

Tricor

Triglide

Abraxane

Drug delivery

CompagnyIndicationNP CompositionApplication

In vivo imaging

Active implants

Pacemaker Fractal electrodes Biotronik (Berlin)Heart failure

Colloidal NP British Biocell and Amersham, BG; Nymox, US

Pregnancy, ovulation, HIV…

Iron NP Dynal/Invitrogen, NO; MiltenylBiotec, DE; Immunicon, US

Immunodiagnostics

In vitro diagnostics

Lateral flow tests

Clinical cell separation

Schering, DE

Advanced Magnetics, US and Guerbet, FR

Advanced Magnetics, Guerbet

Liver cancer

Liver cancer

Imaging of abdominal structures

Iron NP

Iron NP

Iron NP

Resovist

Feridex/Endorem

Gastromark/Lumirem

Dentspley, GB

3M Espe, DE

Heraeus Kulzer, DE

Sybron Dental Specialties, US

Ivoclar Vivadent, LI

Osartis, DE

Aap Implantate, DE

Orthovita, US

Nucryst, US



Dental restoration

Dental repair

Dental repair

Bone defects

Bone defects

Bone defects

Antimicrobial wound care

NP composite

NP composite

NP-containing dental prothesis

NP composite

NP composite

NP-hydroxyapatite

NP-hydroxyapatite

NP-hydroxyapatite

Silver NP

Ceram X duo

Filtek Supreme

Mondial

Premise

Tetric EvoCeram

Ostim

Perossal

Vitoss

Acticoat

Biomaterials

Enzon, US

InterMune, US

Gilead, US and Fujisawa, JP

Gilead

Ortho Biotech, US and Schering-Plough, US

SkyePharma, GB and Enzon

Berna Biotech, CH

Berna Biotech

Zeneus Pharma, GB

QLT, CA and Novartis, CH

Novavax, US

Enzon

Amgen, US

Enzon

Nektar, US and Hoffmann-La Roche, CH

Enzon, Schering-Plough

OSI Pharmaceuticals and Pfizer, US

Nektar, Pfizer

TEVA Pharmaceuticals, IL

Genzyme, US

Elan Drug Delivery and Merck, US

Elan Drug Delivery, WyethPharmaceuticals, US

Elan Drug Delivery, Abbott, US

SkyePharma, First Horizon Pharmaceuticals, US

Abraxis Bioscience, US and AstraZeneca, GB

Fungal infections

Fungal infections

Fungal infections

Kaposi sarcoma

Cancer, Kaposi sarcoma

Cancer

Hepatitis A

Influenza

Breast cancer


Mesopausal therapy

Immunodeficiency disease

Febrile neutropenia

Leukemia

Hepatitis C

Hepatitis C


Acromegaly

Multiple sclerosis

Chronic kidney disease

Antiemetic

Eating disorders

Immunosuppressant

Lipid regulation

Lipid regulation

cancer

Amphotericin B/ lipid complex

Amphotericin B/ lipid colloidal

Liposomal amphotericin B

Liposomal daunorubicin


Liposomal cytarabine

Virosomal hapatitis vaccine

Virosomal influenza vaccine


Liposomal verteporfin

Estradiol in micellar NP

PEG-adenosine deaminase

PEG-G-CSF

PEG-asparaginase

PEG-α-interferon 2a

PEG-α-interferon 2b

Pegylated anti-VEGF aptamer

PEG-HGH

Copplymer of alanine, lysine, glutaminicacid and tyrosine

Crosslinked polyresin

NP aprepitant

NP megesterol acetate

NP megesterol sirolimus

NP fenofibrate

NP fenofibrate

Paclitaxel protein bound NP

Abelcet

Amphotec

Ambisome

DaunoXome

Doxil

Depocyt

Epaxal Berna

Inflexal V Berna

Myocet

Visudyne

Estrasorb

Adagen

Neulasta

Oncaspar

Pegasys

PEG-Intron

Macugen

Somavert

Copaxone

Renagel

Emend

MegaceES

Rapamune

Tricor

Triglide

Abraxane

Drug delivery

CompagnyIndicationNP CompositionApplication

In vivo imaging

Active implants

Pacemaker Fractal electrodes Biotronik (Berlin)Heart failure

Colloidal NP British Biocell and Amersham, BG; Nymox, US

Pregnancy, ovulation, HIV…

Iron NP Dynal/Invitrogen, NO; MiltenylBiotec, DE; Immunicon, US

Immunodiagnostics

- 39 -

Figure 15: Examples of the variety of nanoparticles-based therapeutics in preclinical development (Zhang et al., 2007)

- 40 -

1.6. The future of nanomedicine: health care needs with promising nano-enabled technology impact

An investment strategy to accelerate nanoscience into nano-enabled technology for

medicine and health should reflect both health need (technology pull) as well as science push.

Within this context, science and engineering of nanoscale structures are expected to make

major contributions across the entire medicine and health spectrum ranging from mortality

rate, morbidity an illness imposes on a patient, disease prevalence, and general societal

burden (Murday et al., 2009). The following examples illustrate potential economic and

therapeutic impacts, even if nano-enabled technologies only contribute partial solutions:

• The direct medical cost for cancer in the US for 2007 was about $90 billion. The

National Cancer Institute (NCI) has recognized the importance of nanostructures in the

diagnosis and treatment of cancer in its Alliance for Nanotechnology in Cancer.

Nanotechnology approaches are progressing rapidly in early diagnosis, nano-enabled

contrast agents for in vivo imaging, nanoscale reformulations of chemotherapy agents

for smaller quantities of drug, targeted delivery for smaller side effects, and new

treatments such as nanoparticle-mediated tumour ablation.

• The direct medical cost for diabetes in the US for 2007 was about $116 billion.

Nanotechnology approaches to monitors of glucose levels and production of insulin

are being explored.

• The annual medical care cost for spinal cord injury in the US is about $1.5 billion; the

full costs are estimated as about $10 billion/year. There are promising nano-enabled

approaches to the regeneration of spinal neurons, a capability once thought impossible.

• In the US, so as to remain physically active, approximately 200.000 people receive hip

implants and 300.000 people receive knee implants. The average lifetime of current

orthopedic implants is only 10-15 years; revision surgeries and their recoveries are not

as successful as the first operation. The cost of an implant varies but is roughly

$20.000. Nano-enabled innovations in bone cement and composite structures are

opening new possibilities for improvements in implants.

The 2006 “Nanomedicine: Nanotechnology for Health” publication of the European

Technology Platform Strategic Research Agenda for Nanomedicine presented additional

examples of expected nanomedicine impact. The workshop participants were polled for their

- 41 -

opinion of pressing clinical needs amenable to nano-enabled technology and identified the

following as illustrating the wealth of opportunities:

� Intelligent nanobiomaterials for cell therapy to improve heart function

� Safe and affordable therapeutic strategies to regenerate neural tissues

� Kidney-hollow fiber membranes

� Detoxification implants-correction of metabolic disorders

� Cochlear and retinal implants

� New power source technology for implants

� Repair of articular cartilage and regaining of homeostasis with the joint

� Skin regeneration

� Antimicrobials

� Drug delivery with:

- Targeted pharmacotherapy-tissue/organ

- Therapeutic DNA transfer vectors

- Nanoparticles to carry a therapeutic payload across the blood-brain barrier

- Transfection devices for therapeutic uses.

- Controlled release (especially long term, continuous, and programmed)

- Transient application sonoporation and electroporation

The considerable investment in nanoscience across the world has been leading to new

discoveries (Figure 16). In the second 5 years of the US NNI, but also, as proposed by the

European Union through its European technology platform, there is a growing effort to

identify potential applications for those discoveries and to accelerate their transition into

innovative technology solutions to societal problems.

Research issues and opportunities will embrace the following priorities:

- In vitro and in vivo diagnostics,

- Drugs delivery and therapy,

- Implants and tissues regeneration,

- Biological systems engineering

- Innovations in medical instrumentation and devices

- 42 -

Figure 16 : Future nanomedicine timeline (nanowerk, 2008)

2. Focus on Cancer Diseases

2.1. Cancer

2.1.1. Epidemiology

Cancer remains one of the world’s most devastating diseases, with more than 10

million new cases every year. Cancer is one of the principal causes of death today in the

developed countries: much more than 150 000 cases per year in France. Cancer diseases killed

more than 6.7 million people around the world in 2002, especially in developing countries (by

order relative to the population density): Europe, US, Australia, Asia, Russia and South

America. This tendency is not going to decrease in the next decade. The WHO predicted that

cancer could kill 10.3 million people per year until 2020, especially in newly and non-

industrial countries. It was predicted that 16 million new cases will appear until 2020 (50% of

increase). Figure 17 indicates the mortality at 5 years for the most current cancer.

- 43 -

Figure 17: Cancer statistic with mortality at 5 years (Simon et al., 2007)

2.1.2. Four most frequent cancers

A. Lung cancer

Lung cancer is the second most common cancer with an estimated 1.2 million new

cases being diagnosed every year on a global basis. The “epidemic” of lung cancer mortality

has been identified as a major health issue confronting both developed and developing

countries, with the disease featuring high mortality rates across all countries. Lung cancer is

one of the predominant causes of cancer related deaths in the seven major markets, causing up

to 3 million deaths annually. Lung cancer usually begins in one lung from where it moves to

the lymph nodes and tissues in the other lung. There are two main types of lung cancer, small

cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), the diagnosis of which can

be ascertained by histopathological studies. SCLC is more invasive but comprises only 15-

20% of all diagnosed lung cancer cases, while NSCLC represents an estimated 80-85% of the

classifications and comprises a group of slower growing and therefore more treatable cancers.

B. Colorectal cancer

Colorectal cancer, also called colon cancer or bowel cancer, includes cancerous

growths in the colon, rectum, and appendix. It is an extremely common form of cancer and

overall, the second leading cause of death among cancers in the western world. Colon cancer

is 2.5 times more common than rectal cancer. Approximately 145 300 new cases of colorectal

cancer were diagnosed in the US during 2005. Known hereditary syndromes accounted for at

least 10% of those, and it is likely that more subtle susceptibility factors contributed to the

pathogenesis of many more. Colorectal cancer is a disease that originates in the epithelial cells

lining the gastrointestinal tract. Cancer of the colon occurs when there are abnormal cells that

result in a tumour. Many of the abnormal cells first develop as polyps inside the colon or

rectum, which with time can become cancerous and metastasize. The incidence of colon

25%92%86%44%95%79%27%52%36%54%96%68%Mortalityat 5 years

thyroidoesophaguslungprostateliverstomachbreastkidneybladdercolorectalpancreasovaryCancers

25%92%44%95%79%27%52%36%54%96%68%Mortalityat 5 years 25%92%86%44%95%79%27%52%36%54%96%68%Mortalityat 5 years

thyroidoesophaguslungprostateliverstomachbreastkidneybladdercolorectalpancreasovaryCancers

25%92%44%95%79%27%52%36%54%96%68%Mortalityat 5 years

- 44 -

cancer increases with age, generally occurring in the sixth or seventh decade of life. While

age is a major factor in the incidence of colorectal cancer, sedentary lifestyle, low fiber diets,

diets rich in red and processed meat, excessive alcohol intake, inflammatory bowel

conditions, radiation exposure, and related medical conditions are associated with an

increased risk. A genetic basis for colorectal cancer has been observed in those who have had

a family history of colon cancer or have a family history of familial adenomatous polyposis

(FAP) or hereditary non-polyposis colorectal cancer (HNPCC).

C. Breast cancer

Breast cancer is the most common cancer in women and the leading cause of death for

women aged 40-44. It is second to lung cancer as the leading cause of all cancer deaths in

women and it is estimated to have accounted for 211 240 new cancer diagnoses and 40 410

deaths in 2005 in US (American Cancer Society). The incidence of breast cancer increases

rapidly with age until menopause, after which time it increases more slowly with advancing

years. The majority (over 75%) of breast cancer begins in the (milk) ducts within the breast;

the next most common site is the lobules – the glandular tissue that makes milk. Most breast

cancers are slow-growing and by the time a lump can be felt, it may have been increasing in

size for 5 or 10 years. Breast cancer is a malignant tumour that develops from the

uncontrolled growth of breast tissue cells. These cells can then metastasize to other areas of

the body via the lymph nodes and later spread beyond regional nodes to distant sites in the

body. Though almost entirely found in women, a very small percentage of the cases (under

1%) are discovered in men. Age is a predetermining factor in most incidences of breast

cancer, with sex hormones, genetics and ethnicity being important factors. Notably, women of

European and African origin have been noted to have higher incidence rates of breast cancer.

In the US, one woman in eight will develop breast cancer in her lifetime, but some families

will be affected at even higher rates. Family breast cancer accounts for 5 to 10% for all breast

cancer, and a substantial number of these cases can be linked to mutations in the genes

BRCA1 or BRCA2. Other established risk factors include having no children, delaying first

childbirth, not breastfeeding, early menarche (the first menstrual period), late menopause and

some hormone replacement therapies, as well as diets that feature a high fat and red meat

content. The probability of breast cancer rises with age but breast cancer tends to be more

aggressive when it occurs in younger women, hence there is a need for screening procedures

across all segments of the population. Several studies have also confirmed an association with

- 45 -

alcoholic intake and an increased risk of developing breast cancer. Perceptible decline

mortality has occurred since 1992, most likely due to therapy with tamoxifen and other forms

of chemotherapy, including therapy that targets human epidermal receptor (HER)2/neu. Early

breast cancer usually also has no symptoms and the earlier a tumour is found, the better the

chance of survival. Low rates of mortality are also attributed to the fact that women in the

major markets are more likely to participate in mammography screenings and are more likely

to be diagnosed with breast cancer during the early stages of the disease.

D. Prostate cancer

Prostate cancer is a malignant tumour, or group of cancerous cells, which arises in the

prostate gland, a gland in the male reproductive system located below the urinary bladder and

in front of the rectum. Prostate cancer tends to be slow-growing compared to other cancers,

and as many as 90% of all prostate cancers can remain dormant and clinically unimportant for

decades. As with other cancers, if it is advanced or left untreated in early stages, it can

eventually metastasize through the blood and lymph fluid to other adjacent tissues and organs.

Prostate cancer is the most frequently diagnosed cancer in American men, with an estimated

232 090 cases in 2005. It is also the second leading cause of cancer deaths in males, exceeded

only by lung cancer, with approximately 30 000 deaths from prostate cancer occurring

annually. A plethora of contradictory information exists regarding the cause of prostate

cancer, although it is agreed that the most important factor is age. Prostate cancer occurs

almost exclusively in men over the age of 50, and it is estimated that about 65% men over the

age of 70 have microscopic evidence of prostate cancer. Hereditary factors, diets that have too

much red meat content and environmental factors affecting the male hormones (androgen),

are also implicated in the etiology of prostate cancer.

2.1.3. Molecular features of cancer

A. Cancer generation

Cancer is not a single disease but a wide range of different diseases of which there are

over a hundred types. Cancers can be classified into two broad types: hematological

(malignancies of the blood) or solid tumours. When the body's cells become abnormal and

duplicate out of control a tumour is formed, these may be cancerous/malignant

(spreading/metastasizing) or benign (non-cancerous).

- 46 -

The unique characteristic of cancer is the proliferation of cells of a type different from

the normal complement of the organism. A cancer cell does not obey the complex rules of

architecture and function that govern the usual placement and behaviour of cells within a

tissue. Cancer is distinguished from other abnormal cellular growths that lead to benign

tumours in its characteristic independence from the restrictions present in normal tissues.

Benign tumours expand and compress, but do not attack or invade adjacent tissues. Through

chemical and mechanical means, the cancer cell insinuates itself between and into space of the

normal cells, killing them. Cancer is a genetic disease (Holland et al., 2006). The evolution of

multicellular organisms has involved the development of intercellular communication

required for such processes as embryonic development, tissue differentiation, and as systemic

responses to wounds and infections. Proteomics is increasingly recognized as a method by

which to decipher the molecular mechanisms underlying cancer cell growth and metastasis.

The objectives are twofold: to understand the basic mechanisms of cancer initiation and

progression, and to identify new therapeutic targets. However, cancer is a multifactorial

disease so diverse that a great deal of time and effort will be necessary to define its associated

proteome modifications and to translate these into practical applications for the clinic. Some

information about the activity of oncogenic and tumour suppressor proteins has been obtained

through proteomics.

These complex signaling networks are in large part mediated by growth factors,

cytokines, and hormones. Growth factors mediate their diverse biologic responses by binding

to and activating cell-surface receptors with intrinsic protein kinase-surface receptors with

intrinsic protein kinase activity. To date, more than 50 receptor tyrosine kinases (RTK), which

belong to at least 18 different receptors families, have been identified. Examples of growth

factors: platelet-derived growth factor (PDGF) family, vascular endothelial growth factor

(VEGF), epidermal growth factor (EGF) family, fibroblast growth factor (FGF) family, the

insulin family, hepatocyte growth factor (HGF), neurotrophin growth factor (NGF) family.

The first oncogenes were discovered through the study of retroviruses, ribonucleic acid

(RNA) tumour viruses whose genomes are reverse-transcribed into DNA in infected animal

cells. Proto-oncogenes encode proteins that are involved in the control of cell growth.

Alteration of the structure and/or expression of proto-oncogene can activate them to become

oncogenes capable of inducing in susceptible cells the neoplasmic phenotype. Oncogenes can

be classified into 5 groups based on the functional and biochemical properties of protein

products of their normal counterparts: growth factors, growth factors receptors, signal

transducers, transcription factors and others including programmed cell death regulators. The

- 47 -

activation of oncogenes involves genetic changes to cellular proto-oncogenes. The

consequence of these genetic alterations is to confer a growth advantage to the cell. Four

genetic mechanisms activate oncogenes in human neoplasms: mutation, gene amplification,

chromosome rearrangements and overexpression. The first three mechanisms resulted in

either an alteration of proto-oncogene structure or an increase in proto-oncogene expression.

As proto-oncogenes, approximately 30 tumour suppressor genes have been identified and

definitively implicated in cancer development. Defects in genomic stability genes have also

been implicated in a broad spectrum of human cancers. Like other tumour suppressor genes,

the genomic stability genes are inactivated in human cancers. However, unlike the mutations

in tumour suppressor genes, mutations in genomic stability genes are much more often

inherited in mutant form. For example, DNA mismatch repair gene defects and HNPCC

cancer, genes that contain repetitive DNA sequences, such as microsatellite tracts, might be

expected to be targets of mutation in these cancers (Hamelin et al., 2008).

Due to the large number of cancers, there are, perhaps unsurprisingly, multiple risk

factors implicated in the development of cancer. The influence of these risk factors varies

substantially according to the type of tumor, with the etiology of some cancers being

predominantly influenced by environmental factors, and other cancers by factors related to

infectious disease or hereditary factors.

B. Metastasis

Despite improvements in cancer therapies over the past 50 years, metastatic solid

cancers remain largely incurable, and the survival for patients with these malignancies is often

measured in months. Approximately 30% of cancer patients have clinically detectable

metastasis at the time of initial diagnosis and 40% harbour occult metastasis. Therefore, in

this era of targeted therapies, substantial efforts are being made to identify the optimal target

for each type of cancer in order to succeed to stop dissemination.

Tumour progression towards metastasis is often depicted as a multistage process in

which malignant cells spread from the tumour of origin to colonize distant organs. However,

these basic steps occur in the context of different organs, emerge at different rates and are

clinically managed in different ways depending on the type of cancer.

The genetic basis of tumourigenesis can vary greatly; the steps required for metastasis

are similar for all tumour cells. The detached cells from the primary site use lymphatic vessels

- 48 -

and the bloodstream for transport. Extracellular matrix (ECM) receptors, invasion of the

basal-cell membrane by proteolytic enzymes, entry into the vasculature, and motility factors

of organs are essential for tumour-cell migration. The development of micro-colonies in the

secondary organ is facilitated by growth factors. Angiogenic factors are also essential to

development of metastasis. Invasion and metastasis are the most insidious and life-threatening

aspects of cancer. The capacity for invasion may not be expressed initially or in all tumours.

Once the neoplasm becomes invasive, it can disseminate via the lymphatics and/or vascular

channels that it induces through tumour-stimulated lymphangiogenesis and angiogenesis and

other perturbations of the local environment. Invasion and metastasis kill hosts through two

processes: local invasion and distant organ colonization and injury.

The diverse temporal courses of metastasis in different types of cancer and patient

populations are evident from clinical observations. As the kinetics of disease progression and

distinct physiological barriers can dictate the latency between the infiltrating and colonizing

steps of metastasis, each clinical course has different implications for the organ-selective

evolution of metastatic cell populations. In oestrogen receptor-positive breast cancer, prostate

cancer and ocular melanoma, metastasis might become manifest decades after the removal of

even a small primary malignancy. The absence of immediate clinical relapse implies that

these tumour cells are not fully competent to overtake organs immediately after infiltration. A

protracted period of latency might ensue during which further malignant evolution of the

disseminated cell population, of their microenvironment or of both must occur for

colonization to proceed to invasive carcinoma.

As an end-stage malignant disease, metastatic relapse is often associated with

resistance to therapy. Relapse following systemic treatments might be due to cell intrinsic

mechanisms such as genetic alterations that confer drug resistance following a period of

therapeutic response. Lung adenocarcinomas with epidermal growth factor receptor (EGFR)

mutations respond to EGFR kinase inhibitors but frequently relapse owing to secondary

EGFR mutations that confer resistance. Certain mechanisms of drug resistance might

simultaneously render the tumour more competent for metastasis.

The most frequent location of metastasis for many types of cancers is the first capillary

bed encountered by circulating cells. However, the distribution of metastases varies widely,

depending upon the histologic type and anatomic location of the primary tumour. For example

80% of cancers that spread to bone originate from the breast, prostate, lung, thyroid gland,

and kidney. Others examples of typical sites of metastasis of different solid cancer are

- 49 -

described in Figure 18. Thus, distant organ infiltration and colonization (separated by a

variable period of intervening latency) are general steps that primary tumour cells must

accomplish to metastasize.

Figure 18: Typical site of metastasis for solid tumours (Nguyen et al., 2009)

2.1.4. Anticancer therapy

The instances of diagnosed cancer are expected to continue to rise as a result of

increased life expectancy, aging populations and technological improvements, which are

leading to more sophisticated screening techniques and earlier detection of cancer.

The three primary methods of treating cancer are surgery, radiation therapy, and

pharmaceuticals, each of which can be used alone or in combination, depending on the type of

cancer being treated. The choice of therapy depends upon the location and grade of the

tumour and the stage of the disease, as well as the general state of the patient (performance

status).

Although complete removal of solid tumours from the body is the goal of treatment,

which can sometimes be accomplished through surgery, the tendency for cancers to spread

into the surrounding tissue often makes this difficult. There is no single curative method for

all cancers, but treatment will generally strive to reduce the spread of malignant cell growth.

LungsSarcoma

BonesProstate

Liver and lungsPancreatic

Liver and lungsColorectal

Lungs, brain, skin and liverSkin melanoma

Brain, bones, adrenal gland and liverLung adenocarcinoma

Bone, lungs, liver and brainBreast

Typical sites of metastasisTumor type (solid)

LungsSarcoma

BonesProstate

Liver and lungsPancreatic

Liver and lungsColorectal

Lungs, brain, skin and liverSkin melanoma

Brain, bones, adrenal gland and liverLung adenocarcinoma

Bone, lungs, liver and brainBreast

Typical sites of metastasisTumor type (solid)

- 50 -

A. Surgery

In theory, non-hematological cancers can be cured if entirely removed by

surgery, but this is not always possible. Surgery is the oldest modality of cancer therapy and

still forms the mainstay of treatment in solid tumours. Surgery is increasing combined with

other treatment modalities.When the cancer has metastasized to other sites in the body prior to

surgery, complete surgical excision is usually impossible. In the Halstedian model of cancer

progression, tumours grow locally, and then spread to the lymph nodes, then to the rest of the

body. This has given rise to the popularity of local-only treatments such as surgery for small

cancers. Even small localized tumours are increasingly recognized as possessing metastatic

potential. The goal of the surgery can be either the removal of only the tumour, or the entire

organ.

Surgical procedures currently involve numerous approaches. Many types of surgical

methods for treating cancer and precancerous conditions exist, and investigators continue to

research new methods. Some common types of cancer surgery include:

Cryosurgery: During this type of surgery, very cold material is used, such as liquid

nitrogen spray or a cold probe, to freeze and destroy cancer cells or cells that may become

cancerous, such as irregular cells in a woman's cervix that could become cervical cancer.

Electrosurgery: By applying high-frequency electrical currents, cancer cells are

killed, for example, in the buccal cavity or on the skin.

Laser surgery: Laser surgery, used to treat many types of cancer, uses beams of high-

intensity light to shrink or vaporize cancer cells. In some cases, the heat of the laser

accomplishes this. In other cases, the laser is used to activate a previously administered

chemical that cancer cells absorb. When stimulated by light, the chemical kills the cancer

cells.

Mohs' surgery: Useful for removing cancer from sensitive areas of the skin, such as

near the eye, and for assessing how deep a cancer goes, this method of surgery involves

carefully removing cancer layer by layer with a scalpel.

Laparoscopic surgery: A laparoscope is used to see inside the body without making

large incisions. Instead, several small incisions are made and a tiny camera and surgical tools

are inserted into the body. The surgeon watches a monitor that projects what the camera sees

inside your body. The smaller incisions mean faster recovery and a reduced risk of

complications. Laparoscopic surgery is used in cancer diagnosis, staging, treatment and

symptom relief.

- 51 -

Robotic surgery: In robotic surgery, the surgeon sits away from the operating table

and watches a screen that projects a 3D image of the area being operated on. The surgeon uses

hand controls that tell a robot how to maneuver surgical tools to perform the operation.

Robotic surgery helps the surgeon operate in hard-to-reach areas. But robotic surgical systems

are expensive and require specialized training, so robotic surgery is usually available only in

specialized medical centers.

Natural orifice surgery: Natural orifice surgery is currently being studied as a way to

operate on organs in the abdomen without cutting through the skin. Instead, surgeons pass

surgical tools through a natural orifice, such as your mouth, rectum or vagina. For instance, a

small incision is made in the wall of the stomach and surgical tools pass into the abdominal

cavity in order to take a sample of liver tissue or remove the gallbladder. Natural orifice

surgery is experimental, and few operations have been performed this way. It is expected to

reduce the risk of infection, pain and other complications of surgery.

Cancer surgery continues to evolve. Researchers are investigating other surgical

techniques with a goal of less invasive procedures.

Both, surgery and radiotherapy are essentially local treatments directed at the primary

tumour and any loco-regional disease.

B. Pharmaceuticals

Chemotherapy

Chemotherapy is a systemic treatment that can destroy cancer cells and treat distant

metastases. Chemotherapy has vastly improved the prognosis of many malignant conditions

but it is only curative in a minority of cancers, for example, lymphomas, leukaemias and

testicular cancers. Methotrexate, fluorouracil, doxorubicin, tamoxifen, taxol and gemcitabine

(Figure 19) are the drugs to most indicate to treat cancer.

- 52 -

Figure 19: History of cancer drug availability

In current usage, the term "chemotherapy" usually refers to cytotoxic drugs which

affect rapidly dividing cells in general, in contrast with targeted therapy. Chemotherapy drugs

interfere with cell division in various possible ways, e.g. with the duplication of DNA or the

separation of newly formed chromosomes. Most forms of chemotherapy target all rapidly

dividing cells and are not specific for cancer cells, although some degree of specificity may

come from the inability of many cancer cells to repair DNA damage, while normal cells

generally can. Hence, chemotherapy has the potential to harm healthy tissue, especially those

tissues that have a high replacement rate (e.g. intestinal lining). These cells usually repair

themselves after chemotherapy. Because some drugs work better together than alone, two or

more drugs are often given at the same time, this is called combination chemotherapy. The

effectiveness of chemotherapy is often limited by toxicity to other tissues in the body.

Actively proliferating cells are the most vulnerable to chemotherapy. Non-dividing

cells are killed less effectively by anti-cancer drugs. The cycle (Figure 20) is divided into four

main phases. G1 is the protein synthetic phase; in the S phase, DNA is copied and synthesised

prior to cell division, which occurs following chromosome condensation and segregation in

G2 and M phases. The so-called resting phase, G0, is important. Such cells are not undergoing

cell division and may eventually suffer programmed cell death (apoptosis). However,

following treatment with chemotherapy or radiotherapy some G0 cells may be recruited back

into cycle. The lethal effects of chemotherapeutic agents vary between different phases of the

cell cycle. Cisplatin carboplatin, one of the most utilized drugs for cancer, and the alkylating

agents are lethal throughout the cell cycle, but are particularly active in G2 and at the G1/S

boundary. Antimetabolites such as 5-fluorouracil and methotrexate are most active against

Gemcitabine1990

Taxol1980

Tamoxifen1970

Doxorubicin1960

Fluorouracil1950

Methotrexate1940

ExampleDecade

Gemcitabine1990

Taxol1980

Tamoxifen1970

Doxorubicin1960

Fluorouracil1950

Methotrexate1940

ExampleDecade

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cells in S phase. Etoposide, vinca alkaloids and taxanes are most effective against cells in the

G2/M phases of the cell cycle.

Figure 20: Chemotherapy and the cell cycle (Symonds et al., 2006)

Radiation sensitizers

Chemotherapeutic agents that are highly responsive to ionizing radiation and enhance

the effectiveness of radiation treatment are termed radiation sensitizers. Radiation sensitizers

act in a number of ways to make cancer cells more susceptible to death by radiation than

surrounding normal cells. An ideal radiation sensitizer would reach the tumour in adequate

concentrations and act selectively in the tumour compared with normal tissue. It would have

predictable pharmacokinetics (pK) for timing with radiation treatment and could be

administered with every radiation treatment. The ideal radiation sensitizer would have

minimal toxicity itself and minimal or manageable enhancement of radiation toxicity. The

ideal radiation sensitizer does not exist today.

Targeted therapies

Targeted therapy, which first became available in the late 1990s, has had a significant

impact on the treatment of several types of cancer. This constitutes the use of agents specific

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for the deregulated proteins of cancer cells. Small molecule targeted therapy drugs are

generally inhibitors of enzymatic domains on mutated, overexpressed, or otherwise critical

proteins within the cancer cell. Monoclonal antibody therapy is another strategy in which the

therapeutic agent is an antibody which specifically binds to a protein on the surface of the

cancer cells. Targeted therapy can also involve small peptides as "homing devices" which can

bind to cell surface receptors or affected ECM surrounding the tumour. The trend in the

pharmaceutical industry is to provide more and more drugs or therapeutic tools with a

personalize approach. This is mainly because of the lack of efficacy of a generalized approach

where a population can be seen as one patient with a similar biological profile.

Hormone therapy

It consists to add, block, or remove hormones. To slow or stop the growth of certain

cancers (such as prostate and breast cancer), synthetic hormones or other drugs may be given

to block the body’s natural hormones. Sometimes surgery is needed to remove the gland that

makes a certain hormone. For example, many premenopausal breast cancer patients have their

ovaries removed to eliminate oestrogen from their body. Oestrogen is thought to cause some

types of breast cancer to grow more quickly. These same patients who have tumours classified

as oestrogen-receptor positive can be treated with tamoxifen as an accepted alternative to

ovarian exeresis. Antioestrogen therapy increases survival for some breast cancers.

C. Radiotherapy

Radiotherapy is the use of ionizing radiation to kill cancer cells. Radiation therapy

injures or destroys cells in the target tissue by damaging their genetic material, making it

impossible for these cells to continue to grow and divide. Although radiation damages both

cancer and normal cells, most normal cells can recover from the effects of radiation and

function properly. The goal of radiation therapy is to damage as many cancer cells as possible,

while limiting harm to nearby healthy tissue. The effects of radiation therapy are localised and

confined to the region being treated.

The lethality of radiotherapy is related to its effects on DNA, with the introduction of

single-stranded DNA breaks and, to a lesser extent, double-stranded DNA breaks. DNA

damage arises because absorption of radiation in tissues leads to the immediate production of

ionized atoms, which are raised into excited states. These in turn lead to the formation of

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unstable, short-lived free radicals, which interact with cellular constituents. Hence, it is given

in many fractions, allowing healthy tissue to recover between fractions. Prescribed treatments

typically consist of 25 to 35 fractions, and are administered over periods ranging from a few

days to several weeks. Such fractions are intended to deliver a cumulative dose of radiation

sufficient to kill cancer cells, while allowing healthy tissue to recover sufficiently between

treatments.

Radiation therapy may be used to treat almost every type of solid tumour, including

cancers of the brain, breast, cervix, larynx, lung, pancreas, prostate, soft tissue sarcomas, skin,

stomach or uterus. Radiation dose to each site depends on a number of factors, including the

radiosensitivity of each cancer type and whether there are tissues and organs nearby that may

be damaged by radiation. As with every form of cancer treatment, radiation therapy is not

without its side effects.

For some types of cancer, radiation may be given to areas that do not have evidence of

cancer. This is done to prevent cancer cells from growing in the area receiving the radiation.

This technique is called prophylactic radiation therapy.

Radiation therapy also can be given to help reduce symptoms such as pain from cancer

that has spread to the bones or other parts of the body. This is called palliative radiation

therapy.

Recent advances in radiation therapy technologies have focused on further improving

the ability to target the radiation dose more precisely and to increase the radiation dose in

cancer cells, while minimizing the exposure of healthy tissue.

There are two main types of radiation therapy:

• External radiation is delivered by a machine from outside the body

• Internal radioactive materials are placed in the body near the cancer cells (also called

implant radiation or brachytherapy)

External beam radiation therapy (EBRT)

EBRT creates a radiation beam and aims it at the tumour.

Electromagnetic radiation

The X-ray radiation adequately covers the tumour but minimizes the dose to the non-

tumour normal tissues. Radiation is given in fractions rather than as a single dose.

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Conventional fractionation is 1.8 to 2 Gray (Gy) per day, administered five days a week for

five to seven weeks, depending on the particular clinical situation. While this schedule is

strictly for the convenience of physicians trying to maintain a normal workweek, the

relatively long intervals between doses of radiation may allow cancer cells (as well as normal

cells) to recover and re-grow.

A number of different radiotherapy schedules have been suggested to overcome this

problem. These include hyperfractionation, in which the time between fractions is reduced

from 24 hours to 6 to 8 hours to enhance the toxic effects on tumour cells while still

preserving an adequate time interval for the recovery of normal cells. Continuous

hyperfractionated accelerated radiation therapy (CHART) is an intense schedule of treatment,

in which multiple daily fractions are administered within a short period of time. Clinical

studies have shown benefits of altered fractionation over conventional treatment for several

cancers, including head and neck cancer and non-operable lung cancer.

Three-dimensional conformal radiation therapy (3D-CRT)

3D-CRT is a technique that uses imaging computers to precisely map the volume and

location of a tumour. The patient is fitted with a plastic mold or cast to keep the body part still

so that the radiation can be aimed more accurately from several directions. By aiming the

radiation more precisely at the tumour, it is possible to reduce radiation damage to normal

tissues surrounding the tumour by up to 50 percent.

Intensity modulated radiation therapy (IMRT)

IMRT, involves varying, or modulating, the radiation beam intensity across the

treatment area. This technique attempts to conform the high dose region of the radiation beam

more closely with the shape of the tumour, enabling the delivery of higher doses of radiation

to tumours with a reduced impact on surrounding healthy tissue. Using IMRT, medical

professionals can design a more individualized treatment plan for each patient. This may

result in a higher cancer-control rate and a lower rate of side effects. Adaptive radiation

therapy involves adjusting a patient’s radiation therapy plan between fractions to account for

changes in the patient’s anatomy, the amount and location of the radiation received by the

patient, and the size, shape and location of the tumour. It has been characterized to include as

little as an adjustment to the physical position of the patient relative to the radiation source

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prior to treatment, as occurs during image guided radiation therapy (IGRT), rather than

adjustment to the treatment plan. IGRT consists of an improved type of IMRT device that

combines the functions of diagnostic imaging with a computer guided beam control device to

accurately direct radiation beams to the tumour area and correct error arising due to

movement of patient’s internal organs and tissues. By combining imaging with radiation

treatment, clinicians can adjust the patient’s position relative to the radiation source prior to

each treatment to target the tumour more precisely. Compared to traditional IMRT without

image guidance, accurate image guidance enables clinicians to improve patient outcomes by

concentrating higher doses of radiation at tumours and further reducing the exposure of

healthy tissues to radiation.

Stereotactic body radiation therapy (SBRT)

SBRT is a standard form of treatment for primary and metastatic brain cancer. It is

delivered using a machine called a gamma knife, which uses converging beams of gamma

radiation that meet at a central point within the tumour, where they add up to a very high,

precisely focused dose of radiation in a single fraction. Due to this precision, the cancer can

be located in an area of the brain or spinal cord that might normally be considered inoperable.

CyberKnife®

CyberKnife® is a non-invasive, precise radiation technique that can deliver

concentrated and accurate beams of radiation to any site in the body. This system combines

robotics and advanced image guidance cameras to locate the tumour’s position in the body

and deliver highly focused beams of radiation that converge at the tumour, avoiding normal

tissue. It is a successful method used to treat spinal tumours or tumours at other critical

locations that are not amenable to open surgery or radiation, as well as to treat medically

inoperable patients. It can also be used to treat benign tumours and lesions in a previously

irradiated site, or to boost standard radiotherapy.

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Particulated radiation

Proton beam radiation therapy

This is one of the most precise and sophisticated forms of EBRT available. The

advantage of proton radiation therapy over X-rays is its ability to deliver higher doses of

shaped beams of radiation directly into the tumour while minimizing the dose to normal

tissues. This leads to reduced side effects and improved survival rates. There are

approximately 19 proton treatment centers worldwide.

Internal radioactive materials

Brachytherapy

Brachytherapy or internal radiation therapy uses radiation that is placed very close to

or inside the tumour. The radiation source is usually sealed in a small holder called an

implant. Implants may be in the form of thin wires, catheters, ribbons, capsules, or seeds. The

implant is put directly into the body (NCI).

Radioimmunotherapy (RIT)

Radioimmunotherapy, one of the newest developments in the treatment of non-

Hodgkin's lymphoma, has achieved a high tumour response rate (up to 80 percent) in several

clinical trials. Radioimmunotherapy uses drugs called monoclonal antibodies (mAb), which

have a radioactive isotope attached to them. This is targeted to the surface of a cancer cell,

destroying it. Radioimmunotherapy can be used (in a targeted fashion) to treat single cells that

have spread around the body. Because the radiation does not concentrate in any one area of

the body, radioimmunotherapy does not cause side effects commonly seen with EBRT. The

most significant side effect associated with radioimmunotherapy may be a temporary drop in

white blood cell or platelet count.

Current trends of radiotherapy combinations

Radiation therapy may be used alone or in combination with other cancer treatments,

such as chemotherapy or surgery.

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Radiotherapy and Surgery

Malignant tumours of the brain, head and neck, lung, large parts of the gastro-

intestinal or genito-urinary tracts, and bone or soft tissues are among the most well known

indications for radiosurgical approaches. Improved local and regional control can often be

achieved using radiosurgery. Once the gross disease is resected, radiotherapy can be used to

kill residual tumour cells. Preoperative radiotherapy can make an unresectable tumour

amenable to surgery.

Radiotherapy and chemotherapy

Throughout the past two decades, combinations of radiotherapy with chemotherapy

have yielded encouraging results in patients with locally advanced diseases and for whom the

prognosis remains dismal in terms of local control and distant metastasis. Patients’ benefits

have been observed for malignant epithelial tumours such as head and neck, lung and

gastrointestinal tumours.

D. Other biological anticancer approaches

Immunotherapy-biotherapy

Immnunotherapy is the development of methods to augment and enhance the body’s

natural tendency to defend itself against malignant tumours without damaging healthy tissue.

Immunological and biotherapy treatments which target therapy are a fraction of total cancer

treatment revenues at the current time. Novel treatments such as mAb and vaccines that can

develop resistance to cancer in populations are currently developed. Most therapeutic

vaccines are in phase III trials at this time. To a more limited extent, interleukins (IL) and

interferons are utilized and their use is indicated in tumours such as renal cancer. Other

biotherapies include treatments such as antisense oligonucleotides, protein and polypeptide

growth factors, methylation modifiers, p53 tumour suppressor proteins, regulatory enzymes

and other therapies.

Gene therapy

Gene therapy can defined as the transfer of genetic material into a cell to transiently or

permanently alter the cellular phenotype. Introduction of a nucleic acid or target gene

(transgene) directly into cells is referred to as transfection. Cancer gene therapy is an

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emerging field that was received as one such opportunity to take advantage of the genetic

differences between normal and transformed cells. Gene therapy has dampened interest in

developing approaches to inactivate oncogenes or replace non-functioning tumour suppressor

genes. It designs gene delivery systems (adenoviruses, herpes and non viral), therapeutics

gene, selective gene expression or virus oncolysis (Advani et al., 2006).

Stem cell transplantation

It is a method of replacing immature blood-forming cells that were destroyed by

cancer treatment. The stem cells are given to the person after treatment to help the bone

marrow recover and continue producing healthy blood cells.

E. Other physics-based anticancer approaches

Photodynamic therapy (PDT)

PDT is based on the concept that certain photosensitizers can be localized in neoplastic

tissue, and subsequently, these photosensitizers can be activated with the appropriate

wavelength of light to generate active molecular species such as free radicals and singlet

oxygen (1O2) that are toxic to tumours. Limitations of light penetration make this therapy

most appropriate for small or superficial lesions, such as bladder, oral mucosa, cervical

cancer, Barret’s oesophagus.

Hyperthermia (HT)

HT means elevation of temperature to a supra-physiological level. It is known to cause

direct cytotoxicity and also acts as a radiation and chemosensitizer. HT improves tumour

oxygenation and delivery of novel drug carriers, such as liposomal agents. Recent

developments in the field of gene therapy may also establish a role of HT as a strategy for

targeted, localized induction of gene therapy using the heat shock promoter.

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2.1.5. Energy sources for physics-based anticancer approaches

A. Ionizing radiation sources

Radiotherapy equipments and facilities

The US NCI estimates that, despite of advances in systemic therapy, nearly 50% of

cancer patients in the US are treated using radiation therapy. Radiation therapy is indicated in

approximately 90% of patients with solid tumours, and in the routine medical practice 50 - 60

% of cancer patients are treated with this modality during the disease course. In the US, over

15 million radiation therapy treatments are performed annually in 1.870 radiotherapy centers.

Europe counts 911 radiotherapy centers. Globally more than 5.000.000 patients are treated

with radiotherapy annually. This makes, apart from surgery, radiotherapy as the major method

applied with curative intent for cancer patients (Statistics after J. Sisterson, Particles, no. 17;

1996 and no. 31; 2003). Approximately 90% of patients treated with radiation therapy in the

US and other developed countries receive external beam radiation generated by a device

called a linear accelerator. Linear accelerators have been widely used for radiation therapy for

over 30 years. While radiation therapy is widely available in the US and Western Europe,

many developing countries currently do not have a sufficient number of linear accelerators to

adequately treat their domestic cancer patient populations.

Photons

� X-rays : LINAC (50 KeV � 200 keV ; MeV)

A linear accelerator produces X-rays by slamming a beam of high energy particles

(usually electrons) into a metal target. This gives off X-ray photons in a process called

bremsstrahlung, literally “braking radiation”, that occurs when a charged particle undergoes

acceleration (or deceleration in this case). The energy of the photons can be as high as the

energy of the incoming particles, but the most probable energy is some fraction of that. Most

of the photons are given off in the direction of the incident beam, so this results in a beam of

photons with a spectrum of energies somewhat less than the incident beam energy.

The simplest device for creating X-rays is the X-ray tube. An X-ray tube is simply a

vacuum tube that has a filament on one end that emits electrons and an anode on the other end

to collect them. A high voltage is placed between the filament or cathode and the anode to

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accelerate the electrons. The energy of the electrons is measured in electron volts. One

electron volt is the energy an electron gets when it is accelerated by one volt. If you were to

place a voltage of 1 kilovolt across an X-ray tube, the electrons would have an energy of 1

kilo electron volt (or 1 keV). When the electrons hit the anode, bremsstrahlung photons are

given off. These photons can have energy up to the energy of the incident particles. Therefore,

if 100 kilovolts were placed across the tube, the energy of the electrons would be 100 keV and

the photons could have energy of up to 100 keV. Usually, though, the peak of the spectrum is

at energy of about 1/3 of the incident energy.

Systems like X-ray tubes work well for energies in the kilovolt range, but become

large and cumbersome if used to generate photons in the megavolt range. Since photons with

a higher energy can penetrate further into the patient to treat a deep seated tumour, other

methods of generating radiation that could reach megavolt energies were developed. There are

several types of accelerators that can generate photons with that high of energy, but the one

that is most often used is the linear accelerator. This Figure 21 shows a block diagram of a

typical linear accelerator or linac.

The linear accelerators in use today use microwaves to accelerate electrons. The waves

are generated by a device called a magnetron (you have a smaller version of one in your

microwave oven) or by a microwave amplifier called a klystron. The microwaves then travel

through a waveguide into an accelerator tube. When electrons are injected into the accelerator

tube from an electron gun, they can pick up energy from the waves and be accelerated. The

accelerator tube can be manipulated to give different amounts of energy to the electrons and

therefore different final energies for the beam. Very large accelerators like the Stanford

Linear Accelerator Center can accelerate particles to energies of Giga electron volts. Medical

accelerators are more compact and generate electron beams with energies in the Mega

electron volt (MeV) range. A bending magnet then steers the beam into a metal target to

generate X-rays. The bremsstrahlung process is very inefficient. Only about one tenth of a

percent of the electron beam energy is converted into usable X-rays. Most of the rest is

dissipated in heat in the target. Therefore, the target must be made of something that is very

heat resistant like tungsten and is usually water cooled. Once the X-rays are generated, the

beam is collimated and shaped in the treatment head.

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Figure 21 : Linear accelerator model, (BC Cancer Agency, Vancouver)

� Gamma-Rays (Cobalt, Cesium)

Gamma rays are produced when a neutron transforms to a proton and a beta particle.

The additional proton changes the atom to barium-137. The nucleus ejects the beta particle.

However, the nucleus still has too much energy and ejects a gamma photon (gamma radiation)

to become more stable.

Cesium 137 and Cobalt 60 provides an example of radioactive decay by gamma

radiation. Cobalt 60 is used in many common industrial applications. Large sources of Cobalt

60 are increasingly used for sterilization food or material. The powerful gamma- rays kill

bacteria and other pathogens, without damaging the product.

Electrons: LINAC

Electrons have advantages over photons for some types of treatments. Electrons have a

much shorter range and are therefore more desirable for treatments that are very close to the

skin. It is important to remember though that by removing the target you get rid of the

inefficiency of the bremsstrahlung process. Therefore the electron beam current will have to

be lowered by a factor of 1000 (since the X-ray production of the target is 0.1% efficient) to

have roughly the same dose as an X-ray beam. Electron beams are described in units of MeV,

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as in 6 MeV. Figure 22 shows typical linear energy transfer values for different sort of

radiation sources.

Figure 22: Typical linear energy transfer values

B. Laser light source

The term “laser” stands for Light Amplification by Stimulated Emission of Radiation.

Ordinary light, such as that from a light bulb, has many wavelengths and spreads in all

directions. Laser light, on the other hand, has a specific wavelength. It is focused in a narrow

beam and creates a very high-intensity light. Laser therapy is often given through a flexible

endoscope (a thin, lighted tube used to look at tissues inside the body). The endoscope is

fitted with optical fibbers. It is inserted through an opening in the body, such as the mouth,

nose, anus or vagina. Laser light is then precisely aimed to cut or destroy a tumour.

o For heat generation: HT therapy

Laser-induced interstitial thermotherapy (LITT) (or interstitial laser photocoagulation)

also uses lasers to treat some cancers. LITT is similar to a cancer treatment called HT, which

uses heat to shrink tumours by damaging or killing cancer cells.

During LITT, an optical fibber is inserted into a tumour. Laser light at the tip of the

fibber raises the temperature of the tumour cells and damages or destroys them.

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o For free radical generation: PDT

PDT is another type of cancer treatment that uses lasers. In PDT, certain drugs, called

photosensitizers, are injected into a patient and absorbed by cells all over the patient’s body.

After a couple of days, the agent is found mostly in cancer cells. Laser light is then used to

activate the agent and destroy cancer cells.

C. Ultrasound

o For heat generation: HT

New high intensity focused ultrasound (HIFU) treatment has been implemented for the

treatment of prostate cancer. The procedure uses ultrasound to destroy deep-seated tissue

without affecting the surrounding healthy tissue.

The Sonablate 500 system has approval in Europe for the treatment of prostate

diseases. There are currently 35 units being used in medical institutions around the world.

The delivery of this energy to the tumour small area results in an increase in

temperature to a point where the lipids (fats) in the cell membrane melt and the proteins

denature. A reproducible but small volume of tissue destruction occurs. The distribution of

these target lesions is under the control of the clinician.

o For drug release: drug delivery therapy

Using a combination of polymers that respond to temperature, researchers developed

multifunctional nanoparticle that can image tumours using ultrasound and simultaneously

deliver cell-damaging energy and anticancer drugs to tumours. For instance, ThermoDox®

combines doxorubicin with liposomal technology, which provides heat activated drug

delivery. Celsion’s CEO which developed this product say "It's a liposomal platform that is

unique in that, in a very narrow range of temperatures, the liposome decomposes so it allows

us to be able to target cancers by adding heat at the local site, causing the doxorubicin to be

concentrated at the tumour”.

For example, nanoparticles serve as sensitive and specific targeted contrast and drug

delivery vehicles by binding to specific cell surface markers. Application of acoustic energy

at diagnostic power levels could promote nanoparticle-associated drug delivery by stimulating

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increased interaction between the nanoparticle's lipid layer and the targeted cell's plasma

membrane (Crowder et al., 2005).

D. Magnetic field

Magnetic field is low-energy radiation that comes from the interaction of electric and

magnetic fields. Sources include power lines, electric appliances, radio waves, microwaves,

and others.

o For heat generation: HT

New treatment method is based on a defined energy transmission to biocompatible

nanoparticles in a magnetic field. The resulting high heat production is determined by the

particle type, the frequency of the radiated alternating magnetic field and the magnetic field

intensity.

Therapy is more precisely based on injecting aminosilane-coated iron oxide

superparamagnetic nanoparticles into a tumour. These nanoparticles are then subjected to a

high-frequency alternating magnetic field, causing them to vibrate and produce heat which

then damages or destroys the tumour cells. Depending on the temperature attained within the

tumour, the method may be used either as HT therapy in support of conventional forms of

treatment or by itself as thermoablation for the direct destruction of tumour cells. This

approach, now in clinical trials but not yet commercially available, may be used for many

different types of solid tumours, as fundamentally all tumour cells may be damaged or

destroyed at a certain temperature (Magforce technology).

2.2. Narrowness of the therapeutic index: main concern of anticancer treatments

2.2.1. Limitations of anticancer treatments

Anticancer agents that target the cell cycle and the DNA such as cytotoxics or X-rays

are among the most effective in clinical use and have produced significant increase in the

survival of patients with cancer when used alone or in combination with drugs that have

different mechanisms of actions. They are also extremely toxic and show a narrow therapeutic

window. For these reasons, much effort has been put into modifying current treatments in

terms of technology innovation, as well as understanding the signal-transduction pathways

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that mediate these responses. There is considerable excitement in the cancer field to modify

the therapeutical ratio, aiming at efficacy and safety improvements. Concerning the radiation

therapy, despite of advances in radiation techniques, most commercially available radiation

therapy systems still present significant limitations that restrict clinicians’ ability to provide

the most effective treatment possible. Many patients with cancer either do not respond, or

develop resistance to them.

A. Building the therapeutical window

To get a high probability of cancer disease cure with relatively little risk to healthy

tissues and optimal biodistribution (systemic and loco-regional) of the therapeutical agent

constitutes the main objective when defining anticancer approaches. The success of

treatments in eradicating a tumour depends markedly on the total dose given. Moreover,

genetic variations and co-morbidities determine the individual differences in toxicity reactions

and bioavailability of agents, which ultimately would be a crucial factor for tumour cell death

and incidence of acute and chronic adverse events.

Cancer disease control

The main obstacle to effective treatment is the failure of initial cancer therapy to

eradicate a sufficient number of tumour cells to prevent disease recurrence, which

significantly affects long-term survival. This population of surviving cells following therapy

is called minimal residual disease (MRD), and these cells can go on to find refuge in

protective microenvironments. For example, the presence of bone marrow micrometastasis in

around 30% of patients with breast cancer at the time of diagnosis is a strong predictor of

relapse, despite aggressive treatment, and 15-20% of patients still have disseminated tumour

cells in the bone marrow following treatment.

Resistance to antitumour therapy can be subdivided into two broad categories: de novo

and acquired. Acquired resistance develops over time as a result of sequential genetic changes

that ultimately culminate in complex therapy-resistant phenotypes. Conversely, one form of

de novo drug resistance is environment-mediated drug resistance (EMDR), in which tumour

cells are transiently protected from apoptosis induced by chemotherapy, radiotherapy or

receptor-mediated cell death. This form of drug resistance is rapidly induced by signalling

events that are initiated by factors present in the tumour microenvironment and can be

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subdivided into two categories: soluble factor mediated drug resistance (SFM-DR), which is

induced by cytokines, chemokines and growth factors secreted by fibroblast-like tumour

stroma; and cell adhesion-mediated drug resistance (CAM-DR), which is mediated by the

adhesion of tumour cell integrins to stromal fibroblasts or to components of the ECM, such as

fibronectin, laminin and collagen. As a continuation of this theme, Cordes et al. have coined

the analogous term CAM-RR to refer to cell adhesion mediated resistance to radiotherapy.

The selective pressure of therapy eventually leads to the development of acquired

resistance in these surviving cells and the outgrowth of MRD, causing disease relapse. EMDR

contributes substantially to MRD and to the development of acquired resistance by protecting

tumour cells from therapy until they evolve acquired-resistance phenotypes. Data suggests

that treatment strategies could more efficiently target the less complex CAM-DR phenotype at

earlier stages of disease, before the development of acquired resistance (Meads et al., 2008).

Therapeutic strategies are limited by the tolerance of normal body tissues and this

feature frequently determines the delivery of insufficient dose targeting the tumour. Thus,

initial treatment would create the supporting conditions for acquired resistance.

Pharmacokinetics (pK)

pK study is measuring compound concentrations in all major tissues after drug

administration over a period of time until the elimination phase. It is necessary to monitor the

product concentration long enough to fully describe the behaviour in vivo (usually 3X half-

life; t1/2). The pK profile in the blood can be fitted using various programs to obtain key pK

parameters that quantitatively describe how the body handles the drug. Important parameters

include maximum concentration (Cmax), t1/2, clearance, area under curve (AUC), and mean

resident time (MRT), average time that a molecule of a drug stays in the body. When a drug

formulation shows prolonged blood circulation, an increased t1/2, a reduced clearance, an

increased AUC, and an increased MRT are usually observed. pK data are often used in

deciding the dose and dose regimen for maintaining a desirable blood concentration for

improved therapeutics with minimal side effects. The blood concentration of drugs is highly

correlated with their efficacy and toxicity in most cases, especially for free drugs. However, to

gain insight into how the body handles the product formulation and how the formulation may

affect the efficacy and adverse effects, it is essential to obtain the tissue distribution

information for the agent.

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In fact, high level of accumulation of a product in the target tissue often results in an

enhanced therapeutic effect, and oppositely, a large amount of product distributed to non

target organs may cause unwanted toxicity. pK and tissue distribution studies that allow

investigators to screen formulations for a given drug are extremely important during drug

development. By optimizing the drug formulation, investigators can improve the drug

delivery to the target tissue and reduce drug distribution to the non target tissues to obtain

increased therapeutic activity with minimal side effects.

When a hydrophilic drug is intravenously injected into the body, without much protein

binding, the drug is often quickly eliminated from the blood by renal filtration into the urine.

In the case of a hydrophobic drug, the renal clearance is significantly reduced compared to

that of the hydrophilic drugs due to an increased level of serum protein binding. The

hydrophobic drugs are often transformed into hydrophilic metabolites in the liver and

excreted into the bile or eliminated into the urine.

Figure 23 shows a) The supply of a drug to the tissue will depend on its dose and pK.

b) After leaving the vasculature, flux through tissue can occur through extracellular or trans-

cellular pathways, depending on solubility in water and lipids. Diffusion through water will

vary with size or molecular weight (MW). c) Tissue penetration will be determined by the

balance between delivery and consumption. Cellular metabolism will reduce drug penetration

and build-up within the tissue, and binding and sequestration can increase tissue of a drug but

limit its penetration.

Figure 23: Drug distribution in tissue (Minchinton et al., 2006)

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Toxicology

Although the acute toxicity or side effects of cancer therapy which can cause

symptoms such as nausea, myelosuppression and alopecia is well defined, the late

complications of treatment, including the development of dysplastic or fibrous tissue,

continue to evolve because patients are now surviving longer. However, one of the most

severe side effects following successful cancer therapy is the diagnosis of a second primary

cancer.

Second primary cancers reflect not only the late effects of cancer therapy, but also the

influence of environmental factors that were shared with the initial cancer, such as tobacco

and alcohol use, diet, immune function, hormonal status and environmental exposures.

Despite this, a lack of molecular genetic markers that are specific for therapy-induced cancer

makes it almost impossible to say with absolute confidence whether a second cancer in any

given individual is the result of previous therapy. One possible exception to this is genomic

microsatellite instability (Hamelin et al., 2008), which has been reported with high frequency,

in therapy-related myeloid leukaemia (Allan et al., 2005) and is rare in sporadic myeloid

leukaemia. Increased risks of developing a second cancer have been reported after treatment

with radiotherapy and with structurally and functionally diverse chemotherapy agents,

including alkylating agents, topoisomerase inhibitors and anti-metabolites.

Limited therapeutic window

At present, radiotherapy practice is still essentially pragmatic and based on experience

of what happens to the average variant of a particular type of tumour. Even after 100 years of

clinical radiotherapy, it is still not clear what the optimal fractionating regime should be. The

incidence of severe reactions is dependent on the total radiation dose, the dose per fraction,

the overall treatment time, beam type and energy and the surface area of the skin that is

exposed to radiation. Over time, the exposure of healthy tissue to radiation energy can result

in accumulated damage to healthy tissue in the patient’s body and limit the patient’s future

radiation therapy possibilities. It is well recognized that the addition of chemotherapy to

radiotherapy (chemoradiotherapy) increases the acute side-effect profile of the treatment,

particularly when combined with altered fractionation regimens.

Figure 24 shows the limited therapeutic window with respect to cumulative dose,

through which radiotherapy operates: the more radiosensitive the tumour, the wider the

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therapeutic window; the more radiosensitive the normal tissue; the greater the risk of

permanent damage. Damage to organs such as the spinal cord is of profound importance

owing to the lack of repair of normal tissue and potentially severe sequelae. Limited tolerance

of the small bowel reduces the amount of radiation that can be given to the pancreas and

kidney. In contrast, the cervical mucosa can tolerate very high doses of radiation, allowing for

high doses of radiation to be given locally to enable cure. Other late effects of radiotherapy

include carcinogenicity, mutagenicity and teratogenicity. All can be ascribed to the effect of

ionizing radiation on nuclear DNA, with consequent permanent damage to nuclear material.

Figure 24: Therapeutic window: increase the therapeutic index (Bernier et al., 2004)

B. How to reconciliate the tolerated dose and the curative dose

NanoXrayTM platform is based on a technology that is designed to allow destruction of

cancer cells by inert particles. It thus offers a new treatment weapon that could be used alone,

or in concert with existing anticancer protocols: chemotherapy, surgery, targeted molecules,

and immunotherapy. NanoXrayTM particles are intended to be activated from outside the body

with a conventional X-ray, after injection. And, efficacy is expected to be proportional to the

duration of activation and the number of radiotherapy sessions.

Favourable Unfavourable

A B


A B


A B

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Most solid tumours possess unique pathophysiological characteristics that are not

observed in normal tissues or organs, such as extensive angiogenesis and hence multiple

neovessels with defective vascular architecture, impaired lymphatic drainage/recovery

system, and greatly increased production of a number of permeability mediators. The

phenomenon known as the enhanced permeability and retention (EPR) effect for lipid and

macromolecular agents has been observed to be universal in solid tumours. Enhanced vascular

permeability will enable accumulation of nanoXrayTM nanoparticles in the solid tumour

tissue.

After nanoXrayTM nanoparticles accumulate in the target tissues, a standard X-ray is

applied that is intended to generate a local therapeutic effect, designed to destroy only the

targeted tumour cells. This mechanism suggests total control of the intended therapeutic

effect. In short, usage of nanoXrayTM products is intended to resolve radiation therapy’s

biggest drawback: destruction of healthy tissue and its subsequent deleterious side effects

whit high dose X-ray.

NanoPDT platform is designed initially considering traditional routes of medication

administration such as intravenous (i.v), oral, or intramuscular. These routes use traditional

medication administration systems: needles, syringes, fluted paper cups, i.v bags, and

catheters. It must be noted that these medication administration systems may not always

optimize rapid delivery of the appropriate concentration of compound to the appropriate site,

nor do they necessarily minimize local or systemic toxicity. Thus nanoPDT platform allows a

medication delivery system that concentrates photosensitizer where needed and could reduce

the destruction of surrounding tissues while minimizing side effects.

The method by which the photosensitizer agent is delivered can have a significant effect on its

efficacy. Some drugs have an optimum concentration range within which maximum benefit is

derived, and concentrations above or below this range can be toxic or produce no therapeutic

benefit at all.

To deliver drugs to specific organs, a range of organic systems (e.g., micelles,

liposomes, and polymeric nanoparticles) have been designed. They suffer from limitations,

including poor thermal and chemical stability, and rapid elimination by the immune system.

In contrast, silica particles offer a biocompatible, stable, and stealthy alternative. Pp IX

molecules can be easily encapsulated within silica particles which are administered as i.v

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injection. Enhanced vascular permeability will enable accumulation of Pp IX silica

nanoparticles in the tumour structure. Of the most interest, other challenging issues of PDT

can be addressed including surface segregation, and tumour cell uptake with its subcellular

organelle localization, which ultimately determines tumour destruction.

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PART 2: ACTIVATED NANOPARTICLES TO

TREAT CANCER, A WAY TO ENLARGE THE

THERAPEUTIC WINDOW OF ANTICANCER

APPROACHES

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INTRODUCTION

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1. Nanobiotix commitment: an innovation-based nanomaterial company for

cancer treatment

Use of nanomaterial as the “active product” to bring meaningful clinical benefit to cancer patients

Nanobiotix is an emerging nanomedicine company combining dramatic advances in

nanotechnology and molecular biology to develop nanomaterials that are expected to be

turned “on” and “off” outside the body to selectively and safely treat a variety of cancers.

Nanobiotix wants to play the seminal role in making the cancer treatments more effective, less

deadly to healthy tissues.

As a general trend, clinical applications exploit nanotechnology to improve the quality

and sensitivity of a wide variety of different technologies in order to create new approaches

for drug delivery and imaging, with novel targeted agents too. So far, most applications of

nanotechnology in medicine have an enabling function in many different areas.

Unlike, Nanobiotix has explored other fields, enabling the use of nanomaterials as the “active

product” with therapeutic purposes. The company has successfully integrated two worlds, the

promising nanotechnology industry and medicine.

To create and develop nanomaterial as being the “active product” for cancer therapy,

Nanobiotix has built on a technology founded on two major axes: the key understanding of

the biological mechanisms and the ability to elaborate complex structures at the nanometer

scale.

Based on the existing technology and preclinical results, Nanobiotix is developing

different nanomedicine programs that will sustain its pipeline and will expand the uses of

nanoparticles in medicine.

• nanoXrayTM platform which is based on crystalline nanoparticles to be activated by

X-ray

• nanoPDT platform which is based on photosensitizer nanocarriers particles for

treatment of cancer

• nanoMag platform which is based on magnetic particles for treatment and diagnostic

of cancer

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• activated drug delivery platform which uses nanomaterials based on different

modalities already in use in hospitals such as drug-delivery systems (DDS) that can be

externally triggered

NanoXray™ platform lead product NBTXR3 nanoparticle is a non-drug agent able on its

own to kill tumour cells. NBTXR3 nanoparticles can be injected into cancer patients at the

tumour site and taken up by cancer cells. Then patients would undergo a standard X-ray

procedure that would “switch on” the destructive capability of the NBTXR3 nanoparticles,

causing the cancer cells death. NanoXrayTM, in short, allows for the controlled generation of

physical reactions in targeted cells triggered by the application of an external energy source –

a standard X-ray.

NBTXR3 clearly is intended to fight the most deadly cancers (Figure 25).

Figure 25 : NBTXR3 intended initial indications

NanoPDT platform offers the possibility of impacting the pK of photosensitizers such

as Pp IX and others, but as important as this improvement, nanocarriers introduce the

possibility of controlling sub-cellular availability. Efficient PDT may increase the choice of

anticancer modalities, opening the possibility of a “new line of local treatment”, so far highly

limited by the marked changes induced by the radiotherapy in the irradiated tissues. To

improve the tumour bioavailability, to reduce photosensitizer accumulation in the skin or to

render it “cryptic”, to differentially deliver the nanocarriers to cell organelles constitute the

major effects of nanoPDT products.

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The design and mode of action of NBTXR3 – but also of other nanomaterials currently

in Nanobiotix pipeline – is based on a physical effect and not on a biological effect. This

design undoubtedly offers a rupture toward current therapies which focus their efforts on

developing targeted therapeutics agents such as mAb, issued from the characterisation of

molecular features of cancer cells. This is an exciting field, source of new candidates for

anticancer treatment which needs a profound knowledge of any cancer type, tissue origin,

model, stage and genetic background.

Nanobiotix technology is thought very differently as its nanomaterials present a high

level of uncoupling between the therapeutic core of the nanoparticles, which is responsible for

the therapeutic effect, and the coating which confers to the material its specificity for action.

Such approach allows for a much broader range of application in oncology regardless the

disturbed pathways causing uncontrolled growth, proliferation and lack of determined

differentiation and programmed death.

Indeed, the development of nanomedicine products which act by physical mechanism

of action is a new way of thinking medicine. The rational for design comes from the physic

and chemistry, allowing a global approach of cancer therapy. It is not intended to treat

diseases through a specific signaling pathway which act on a single disease target. In contrast

it is intended to deliver the therapeutic effect through a non specific pathway which works

against cancers of a given type or even cancers of many types, a physically-induced pathway

level. As opposed to the development of new drugs for therapy, this very unique concept,

translated to the clinic, is a new weapon in the fight against cancer.

Today, numerous cancers are difficult to treat due to heavy secondary effects arising

when trying to efficiently treat the diseases. Two axes are achievable to solve this issue: either

to reduce the toxicity of the treatment, or to increase the tumour control or its destruction.

Nanobiotix aims at playing on both axes and to reach an unprecedented benefit over risk ratio

combining major changes in efficacy and allowing possibility of anticancer treatment to

patient populations currently excluded from therapies due to their co-morbidities.

Its intention is clearly to break the classical correlation which exists between therapeutic

efficacy and corresponding toxicity. Moreover, the principle of occupancy intervention at the

sub-cellular level to challenge the narrowness of the therapeutic index is a new paradigm.

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2. Nanomaterial and energy interaction: concept and rational for design of

nanomaterial

When thinking about devices, currently used in the clinic for diagnosis and therapy,

which produce external energy sources, one can easily list the following: radiation emitting

devices, MRI, laser and ultrasound. Those external energy sources interact with matter when

passing through, generating a significant change in the surrounding media which triggers the

visualization and/or the treatment of the area of interest.

A step further would be to ask: how does it work? To answer the question, one should

consider the interaction between the energy delivered and the biological media it traverses.

Ionizing down to infrared radiation: the electromagnetic spectrum (Figure 26)

Ionizing radiations are capable of breaking the DNA molecule of the cell, thereby

preventing cells from growing and dividing. This effect is mainly due to damages created by

electrons and/or high energy photons (energy higher than 2 KeV) emitted after ionization.

The term “Ionizing radiations” refers to highly-energetic particles or waves that can detach

(ionize) at least one electron from an atom or molecule. Ionizing ability depends on the

energy of individual particles or waves, and not on their number. A large flood of particles or

waves will not, in the most-common situations, cause ionization if the individual particles or

waves are insufficiently energetic. The ability of light waves (photons) to ionize an atom or

molecule varies across the electromagnetic spectrum. X-rays and gamma-rays will ionize

almost any molecule or atom; far ultraviolet (UV) light will ionize many atoms and

molecules; near UV and visible light are ionizing very few molecules; infra-red (IR),

microwaves and radio waves are non-ionizing radiations. However, IR is able to excite

molecules or atoms.

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Figure 26: The electromagnetic spectrum

Magnetic field

Magnetic field, generated by MRI devices, interacts with the nucleus of hydrogen

atom. Hydrogen is the most abundant element in biological systems and proton in the

hydrogen nucleus acts as a tiny magnet with a non zero magnet moment. When placed in a

large magnetic field, the randomly oriented magnetic vectors of each proton tend to line up

either with (parallel) or against (antiparallel) the direction of the magnetic field. With time,

more protons in any given tissue will align parallel to the magnetic field and a single vector of

magnetization can be described for the sum or ensemble of protons. Each distinct tissue type

has a characteristic rate for its protons to achieve such a magnetization. This magnetization

reflects the ability of each tissue’s protons to exchange energy with the molecular

environment. By adding energy in the form of a radio frequency (RF) pulse, energy which

corresponds to the energy difference between the parallel and antiparallel state, the overall

magnetic vector is abolished as the protons suddenly begin to switch between the low energy

(parallel) and high energy (antiparallel) state in unison. As they all drop together from the

high energy state to the low one, the combined energy they give up is released in the form of

an RF signal, which can be detected for imaging (Brant-Zawadzki et al., 1985).

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Ultrasound

Sound is a physical phenomenon which transfers energy from one point to another. In

this respect, it is similar to radiation. It differs from radiation, however, in that sound can pass

only through matter and not through a vacuum as radiation can. This is because sound waves

are actually vibrations passing through a material. One of the most significant characteristics

of sound is its frequency, which is the rate at which the sound source and the material

vibrate. The human ear cannot hear or respond to all sound frequencies. The range of

frequencies that can be heard by a normal young adult is from approximately 20 Hz to 20.000

Hz (20 kHz). Ultrasound has a frequency above this range. Frequencies in the range of 2

MHz (million cycles per second) to 20 MHz are used in diagnostic ultrasound (Figure 27).

When a beam of ultrasound pulses is passed into a body, several things happen. Most of the

ultrasound energy is absorbed and the beam is attenuated. This is undesirable and does not

contribute to the formation of an image like in X-ray imaging. Some of the pulses will be

reflected by internal body structures and send echoes back to the surface where they are

collected and used to form the image. Echoes are produced by surfaces or boundaries

between two different types of tissues. Therefore, the general ultrasound image is a display of

structures or reflecting surfaces in the body that produce echoes.

Figure 27: The ultrasound spectrum

Could it be then possible, using nanomaterials in combination with those external

energy sources, to improve either the diagnostic or the treatment of a disease, by increasing

either the sensitivity and/or the efficacy of the current effect?

Coming back to nanotechnology, an approach is to consider which type of

nanomaterial could be used to interact with the delivered energy source to potentially improve

the way its energy is exploited. The nanomaterial should first achieve specific characteristics

which may allow its interaction with the energy source. Subsequently the nanomaterial-

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energy interaction should trigger a biological response in the desired way. Some nanoparticles

with market approval or in clinical phase already present such properties. Contrast agents

developed for MRI, are typically superparamagnetic nanoparticles which enhance locally

MRI signal, hence the diagnostic, providing an efficient biodistribution of the nanoparticles

[Resovist®, Schering DE; Feridex®, Advanced Magnetic Industries, Inc. US]. Laser light

sources, used to activate metallic nanoparticles such as gold nanoparticles, cause absorption

of energy by the nanoparticles and a subsequent generation of heat which locally induce cells

and tissues destruction (Maltzahn et al., 2009; O’Neal et al., 2004).

Hence nanotechnology is able to design nanomaterials that are interacting with

external energy sources to tackle unmet needs in the clinic. A key step in the development of

those nanomaterials will be their safe administration into the human body at the right place, at

the right concentration and at the right time.

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3. Nanomaterials and biological media interactions: a necessary

understanding to bring the concept of nanomaterial as “active product” to

the clinic

3.1. The “nano-bio” interface: importance of the protein-corona concept

Knowledge of the nanomaterial-biological interactions is the foundation to understand

how engineered nanomaterials and biological entities will communicate and ultimately how

nanomaterials and biology, will respond to each other.

At the frontier between two scientific worlds – the molecular and cellular biology and

the physic and chemistry – scientists stand at the very beginning of an appreciation of

nanomaterials-biological entities interactions. The outcome will support the design of

optimized nanosystems which need to reach an exquisite level of interaction with their

biological environment for efficient and safe use of nanomaterials in biology.

When nanoparticles enter a biological fluid, they are coated with proteins that may

undergo conformational changes, leading to exposure of new epitopes, altered function and/or

avidity effects. Further, the interactive nanoparticle surface might be pre-bound to chemical

substances that reflect its prior history and could influence its protein adsorption kinetics.

While within the biomaterials field the role of adsorbed molecules in cellular responses is

acknowledged, there are several new issues at stake where nanoparticles are concerned. The

highly selective protein adsorption, added to the fact that particles can reach subcellular

locations, results in significant new potential impacts for nanoparticles on protein interactions

and cellular behavior.

It is a universal rule of materials in biology that a material is always covered by

proteins immediately upon contact with a physiological environment, and it is believed that

this phenomenon will also be key to understanding much of the bionanoscience world. Lynch

et al, 2007 have recently argued that the effective unit of interest in the cell-nanomaterial

interaction is not the nanoparticle per se, but the particle and its ‘corona’ of more or less

strongly associated proteins from serum or other body fluids (Lynch et al., 2007 ; Cerdervall

et al., 2007). It is important to understand, though, that it is not just the composition and

organization of this protein layer, but the exchange times of the proteins on the nanoparticles

that are ‘read’ by living cells. In essence, Lynch et al., 2007 expect a huge range of

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equilibrium constants (one for each protein) representing the quite different (and competitive)

binding mechanisms present. This means that they see the proteins associated with a particle

as a ‘corona’, rather than a solid fixed layer (Figure 28).

The composition of the protein corona at any given time will be determined by the

concentrations of the over 3700 proteins in plasma (Muthusamy et al., 2005), and the kinetic

on and off rates (or equilibrium binding constants) of each protein for the particular

nanoparticle. This corona may not immediately reach equilibrium when exposed to a

biological fluid. Proteins with high concentrations and high association rate constants will

initially occupy the nanoparticle surface, but may also dissociate quickly to be replaced by

proteins of lower concentration, slower exchange, and higher affinity (Cedervall et al., 2007).

Thus the protein corona is the biological identity of a nanoparticle, as it is what the cell

‘sees’ and interacts with. The exchange processes may also be important when particles

redistribute from one compartment or organ to another, such as upon uptake into cells from

the bloodstream, or upon transport from the cytosol to the nucleus.

Because the protein corona could shape the cellular interactions with nanomaterials, a

key question is whether such protein interactions depend on particle composition. The nature

of the particle surface (for example its hydrophobicity, size, radius of curvature, charge,

coatings that exert steric or electrostatic effects) will control which biomolecules interact with

the particles – and hence mediate their access to cells. Several proteins are known to form

transient complexes with nanoparticles. Complement and immunoglobulin binding leads to

particles opsonisation; that is, it promotes receptor-mediated phagocytosis. It is generally

perceived that plasma protein binding is important in determining the in vivo organ

distribution and clearance of carrier particles from the circulation. Proteins and organic

substances increase the dissolution rate of particles of ZnO, CdSe, iron oxides, aluminium

oxides and hydroxides. Studying the reverse effect of particles on protein is important for

understanding potential biological injury due to such changes as protein fibrillation, exposure

of new antigenic epitopes and loss of function such as enzymatic activity.

Probing these various interfaces allows the development of predictive relationship

between structure and activity. This knowledge is important from the perspective of a safe

and efficient use of nanomaterials (Nel et al., 2009).

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Figure 28: The corona constitutes a primary nano-bio interface that determines the fate of the

nanoparticle and can cause deleterious effects on the interactive proteins. Pre-existing or initial material characteristics contribute to the formation of the corona in a biological environment.

Characteristic protein attachment/detachment rates, competitive binding interactions, steric hindrance by detergents and adsorbed polymers, and the protein profile of the body fluid lead to dynamic

changes in the corona. The corona can change when particles move from one biological compartment to another (Nel et al., 2009).

3.2. The nanomaterial delivery at the right time, at the right place and at the right concentration

Due to the growing importance of nano-biotechnology and its enormous potential to

improve drug delivery, the main focus in next years should be on nanomedicine and in

particular on nanomaterial delivery. Nano-scale drug carriers play an important role in the

safe and efficient delivery of active compounds to their intended site of action, but there are

still challenges that need to be solved, like the targeting of specific cell tissues or organs and

to overcome biological barriers such as the skin, intestinal, respiratory mucosa or blood-brain-

barrier, but also the cells and at the subcellular level, the organelles.

Safe nanoparticles administration, with minimum side effects, and effective

bioavailability are the main concerns of current medical cares. Bioavailability refers to the

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presence of nanoparticles at the right concentration and the right anatomical area. The ability

to alter the pK and pharmacodynamic of a product in the body determines its efficacy and

safety. Ultimately, the aim is to deliver nanoparticles at the right time, the right place and

right concentration.

3.2.1. Major obstacles to reach the target: tissues, cells and organelles

A. Tissues

In general, biological compartments act as barriers to the passage of nanosized

materials, and there are several barriers to consider. Firstly, the epithelium acts as a general

barrier to prevent ingress of materials into the body. The next barrier to nanoparticle

distribution is the vascular endothelium. Typically most endothelium is continuous with tight

junctions between the endothelial cells and the underlying basement membrane. The presence

of tight junctions means that the gap between endothelial cells is 2 nm, thus preventing most

nanoparticles from exiting the circulation by passage between the cells. In some specialized

tissue like liver, the endothelium is fenestrated, thus allowing materials up to 100 nm to pass

from the endothelium to the underlying parenchymal cells. In other tissues like spleen, the

endothelium is discontinuous, having larger fenestrations, and also lacks basement membrane,

allowing exit of very large particles. In contrast, in the brain the endothelial junctions are

particularly tight and effective, reducing still further the ability of materials to pass through.

This allows the passage of macromolecules through the endothelium, and is thought to be size

dependent, so larger macromolecules pass through less easily than smaller molecules. Also,

under certain disease conditions, e.g. inflammation, the endothelium can become leaky,

allowing a greater exit of particulate materials (Shrivastava et al., 2008).

In fact, hepatic filtration, tissue extravasation, tissue diffusion, kidney filtration play a

crucial role on particles diffusion. Endothelia composing the blood vessels have been

classified as continuous, fenestrated or discontinuous depending on their morphological

features. The continuous endothelium appears in arteries, vessels and the lungs. Fenestrated

endothelium appears in glands, digestive mucosa and kidney. Fenestrations have pores of

approximately 60 nm. Discontinuous endothelium is a characteristic of the liver (50-100 nm)

and bone marrow (Alexis et al., 2008).

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B. Cells

Every cell is locked into a membrane, an envelope defender of 8-12 nm, which bounds

the cellular compartment and separates it from the surrounding environment. The membrane

plays at the same time the role of a filter and a way of transport. On one hand, it controls the

entry of the nutritive substances and the exit of the cellular wastes and, on the other hand, it

creates an internal environment different from the external environment. It has another

important function which is to create and maintain intracellular concentration of specific ions,

which are atoms or group of atoms carrying an electric load. The cellular membrane acts also

as sensor of external signals, giving to the cells the possibility to answer to the various stimuli

they received.

According to the fluid mosaic model of Singer and Nicolson, the biological

membranes can be considered as a two-dimensional liquid where all lipids and protein

molecules diffuse more or less freely (Singer et al., 1972). This picture may be valid in the

space scale of 10 nm. However, the plasma membrane contains different structures or

domains that can be classified as (a) protein-protein complexes; (b) lipid rafts, (c) pickets and

fences formed by the actin-based cytoskeleton; and (d) large stable structures, such as

synapses or desmosomes.

The permeability of membranes is the ease of molecules to pass through. Permeability

depends mainly on the electric charge of the molecule and to a lesser extent the molar mass of

the molecule. Electrically-neutral and small molecules pass the membrane easier than charged

and large ones.

The inability of charged molecules to pass through the cell membrane results in pH

parturition of substances throughout the fluid compartment of the body. pH parturition is the

tendency for acid molecules to accumulate in basic fluid compartments, and basic molecules

to accumulate in acidic compartments. The reason is that acid molecules become positively

charged in basic fluids, since they donate a proton. On the other hand, basic molecules

become negatively charged in acid fluids, since they receive a proton. Since electric charge

decreases the membrane permeability of substances, once an acid molecule enters a basic

fluid and becomes electrically charged, it cannot easily escape that compartment and therefore

accumulates. Same effect is observed with basic molecules.

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C. Organelles

The physicochemical properties of drugs (for example, MW, shape, charge and

aqueous solubility) determine their localization into the cell.

1- The mitochondrion has been shown to be a critical target in PDT. Lipophilic

porphyrins have demonstrated intimate intracellular association with mitochondrial

membranes, whilst cationic compounds such as rhodamines and cyanines may accumulate in

these organelles due to mitochondrial membrane potential. The inter-membrane space of

mitochondria contains a number of soluble molecules whose release from the organelle to the

cytosol or the nucleus induces cell death. Thus, molecules that directly trigger mitochondria

membrane permeabilisation are efficient cytotoxic drugs. Zhao and his colleagues (2009)

have synthesized 25-30 nm silicon phthalocyanine 4 (Pc4), a photosensitizer for PDT

encapsulated in silica nanoparticles. Cell viability measurement demonstrated that Pc4

nanoparticles was more phototoxic on non-pigmented human melanoma A375 or pigmented

mouse melanoma B16F10 melanoma cells than free Pc4. These nanoparticles were localized

both in the mitochondria and lysosomes compartments.

2- The nucleus is a membrane-bound organelle and is surrounded by a double

membrane. It communicates with the surrounding cytosol via numerous nuclear pores.

The nuclear envelope (NE) is a double membrane, each constituted by lipid bilayer,

which encloses the genetic material in eukaryotic cells. The NE also serves as the physical

barrier, separating the contents of the nucleus (DNA in particular) from the cytosol

(cytoplasm). The outer membrane is continuous with the rough endoplasmic reticulum (ER)

while the inner nuclear membrane is the primary residence of several inner nuclear membrane

proteins. The outer and inner nuclear membranes are fused at the site of nuclear pore complex

insertion. Many nuclear pores are inserted in the nuclear envelope, which facilitate and

regulate the exchange of materials (proteins such as transcription factors, and RNA) between

the nucleus and the cytoplasm. At present, it is admitted that nanoparticles can not cross the

nuclear membrane, but the presence of certain type of nanoparticles (less than 4 nm) has been

described into the nucleus (Tsoli et al., 2005; Gu et al., 2009).

Futhermore, others groups have reported the Tat peptide-mediated import of different

cargos into cells nucleus, including dye-labeled streptavidin protein, 43 and 90 nm fluorescent

beads, as well as ~20 nm quantum dot (QD) for kinetic measurements. Surprisingly, Tat

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peptide was able to import 90 nm beads into the nuclei of digitonin-permeabilized cells,

suggesting that their interaction with the NE follows a mechanism different from that of NLS

(Nuclear Localization Signal). The import kinetics was quantified using Tat peptide-

conjugated QD, yielding a kinetic constant of 0.0085 s−1. The results suggest that, compared

with NLS, Tat peptide-mediated nuclear import is faster, follows a different pathway, and is

capable of importing large nanoparticles. These results have significant implications for the

development of new approaches for delivery of cargo into the nuclei of living cells (De

Lafuente et al., 2005; Nitin et al., 2009)

3- The Golgi apparatus is a membrane-bound structure with a single membrane. It is

actually a stack of membrane-bound vesicles that are important in packaging macromolecules

for transport elsewhere in the cell. The stack of larger vesicles is surrounded by numerous

smaller vesicles containing those packaged macromolecules. The enzymatic or hormonal

contents of lysosomes, peroxisomes and secretory vesicles are packaged in membrane-bound

vesicles at the periphery of the Golgi apparatus.

4- The Golgi complex sorts the lysosomal enzyme in the Trans region. Lysosomal

morphology varies with the state of the cell and its degree of degradative activity. Lysosomes

carry hydrolases that degrade nucleotides, proteins, lipids, phospholipids, and also remove

carbohydrate, sulfate, or phosphate groups from molecules. The hydrolases are active at an

acid pH which is fortunate because if they leak out of the lysosome, they are not likely to

produce damage (at pH 7.2) unless the cell has become acidic. They degrade the products of

ingestion, such as the bacteria that has been taken in by phagocytosis. Lysosomes also

degrade worn out organelles such as mitochondria. A third function for lysosomes is to

handle the products of receptor-mediated endocytosis such as the receptor, ligand and

associated membrane. In this case, the early coalescence of vesicles, bringing in the receptor

and ligand, produces an endosome. Then, the introduction of lysosomal enzymes and the

lower pH cause release and degradation of the contents. This can be used for recycling of the

receptor and other membrane components.

5- There are 2 regions of the ER that differ in both structure and function. One region

is called rough ER because it has ribosomes attached to the cytoplasmic side of the

membrane. The other region is called smooth ER because it lacks attached ribosomes.

Typically, the smooth ER is a tubule network and the rough ER is a series of flattened sacs.

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The smooth ER has a wide range of functions including carbohydrate and lipid synthesis. It

serves as a transitional area for vesicles that transport ER products to various destinations.

The space inside of the ER is called the lumen. The ER is very extensive and is continuous

with the nuclear envelope. Since the ER is connected with the nuclear envelope, the lumen of

the ER and the space inside the NE are part of the same compartment. Chang and his

colleagues (2008) showed that 13 nm gold nanoparticles were co-localized with ER and Golgi

apparatus in B16F10 melanoma cells following 18 hours of incubation with cells.

6- The cytoskeleton is a dynamic 3D filamentous structure within the cytoplasm.

Cellular cytoplasm is dominated by the viscoelastic network of the cytoskeletal lattice,

comprising microfilaments (actin filaments and contractile actomyosin filaments),

microtubules and intermediate filaments. The cytoskeletal lattice is directly responsible for

determining cell shape, generating mechanical forces, resisting externally imposed forces, and

transducing extracellular biochemical and mechanical stimuli to the cytoplasm. Cytoskeletal

dynamics enable the remodeling that is necessary for cell migration and chemotaxis which

underlies tissue development, the inflammatory response, and tumour invasion and metastasis.

Microfilaments are 3-6 nm in diameter, and are composed mostly of the contractile protein

actin, the most abundant cellular protein. Microfilaments are responsible for the cellular

movements of gliding, contraction, and cytokinesis (division of the cytoplasm). For instance,

Zhang et al., 2009 tested quantum dots (QD) nanoparticles (CdSe/ZnS core/shell structure).

They showed that QD nanoparticles with a carboxylic acid surface coating were recognized

by lipid rafts but not by clathrin or caveolae in human epidermal keratinocytes (HEKs). QD

nanoparticles were internalized into early endosomes and then transferred to late endosomes

or lysosomes. QD nanoparticles induced more actin filaments formation in the cytoplasm.

3.2.2. Nanomaterial design for optimized biodistribution and

intracellular trafficking: the importance of the nanomaterial size, shape

and surface coating

Numerous studies demonstrated the influence of nanoparticles size, shape and surface

coating on their biodistribution. Moreover the interactions of nanoparticles with cells are

known to be strongly influenced by these parameters which modulated cellular internalization

and intracellular trafficking.

- 91 -

A. Nanomaterial size

Particles size plays always a role in how the body responds to, distributes and

eliminates materials. By analysis of pegylated nanoparticles uptake by murine macrophages

and blood clearance kinetics, blood clearance of the smaller nanoparticles was twice as slow

as that of larger nanoparticles (Alexis et al., 2008). Also, biological interactions with

nanoparticles could decrease or cancel particles efficacy.

Particle size can also affect the mode of endocytic pathway, cellular uptake and the

efficiency of particle processing in the endocytic pathway (Lanone et al., 2006). In vitro

studies on murine melanoma cell lines B16F10 have demonstrated, with latex nanospheres, a

slower uptake for particles size superior to 200 nm relative to the small ones (50 and 100 nm)

(Rejman et al., 2004). Decreasing the size also leads to an exponential increase of surface

area relative to volume, thus making nanomaterials surface more reactive itself and to

surrounding environment. The size of the nanoparticles was shown to have a substantial effect

on the proteins adsorption.

Increase nanoparticles uptake into certain tissues may lead accumulation, where they

may interfere with the critical functions (Lanone et al., 2006; Kreyling et al., 2006). As

partitioning across membranes is not possible for macromolecules, entry into cells is largely

governed by biological mechanisms of endocytosis (Shrivastava et al., 2008). These include

the uptake of large particles by phagocytosis, performed by specialized cells such as

macrophages and neutrophils, and a variety of other endocytic processes at a smaller scale.

The best known of these, clathrin-mediated endocytosis, involves a vesicle of a defined size

of 100 nm, and can promote uptake through a number of different routes through the cell. A

second mechanism, more recently described, is potocytosis involving caveolae, flask-shaped

vesicles of 70 nm diameters (Shrivastava et al., 2008). These routes of uptake, which include

macropinocytosis, can potentially allow capture of materials up to 300 nm in diameter. All

these endocytic routes of uptake involve delivery of material into a subcellular compartment,

the endosome, which is still separated from the cell cytoplasm by a membrane.

More recently, uptake of small hydrophobic particles directly into the cytoplasm of

cells has been reported in the literature, and called patocytosis (Garnett et al., 2006).

Patocytosis is a unique macrophage endocytosis pathway in which aggregated low density

lipoproteins (LDL) and microcrystalline cholesterol induce and enter a labyrinth of

membrane-bound compartments that remain connected to the cell surface. This process has

been characterized as size dependent, not occurring for particles larger than 500 nm and

- 92 -

associated with the process of capping, in which the particles become trapped in membrane

folds which remain open to the intracellular space.

B. Nanomaterial shape

Particle shape varies from perfect spheres, such as droplets, to fibers and more

recently nanotubes. Particle shape is known to affect the aerodynamic, as well as the diffusive

behaviour of the particles. Particles shape also affects phagocytosis by macrophages. Alveolar

macrophages with diameters in the size range of 10-15 µm are not able to completely engulf

and clear fiber nanoparticles from the alveolar epithelium.

Regarding inorganic metallic or oxide nanoparticles, spherical shape are commonly

admitted as having the best conformation for cell uptake. In particular, Chithrani et al., 2006,

have shown that spherical gold nanoparticles present the highest cell uptake when compared

with rod-shaped gold nanoparticles.

Unlike, Gratton et al., 2007 reported on the internalization of specially designed,

monodisperse hydrogel particles into HeLa cells as a function of size, shape, and surface

charge. They employed a top-down particle fabrication technique called PRINT that is able to

generate uniform population of organic micro- and nanoparticles with complete control of

size, shape, and surface chemistry. Their findings suggested that HeLa cells readily internalize

non spherical particles with dimensions as large as 3 �m by using several different

mechanisms of endocytosis. Moreover, it was found that rod-like particles enjoy an

appreciable advantage when they come to internalization rates, reminiscent of the advantage

that many rod-like bacteria have for internalization in nonphagocytic cells.

C. Nanomaterials surface coating: rational to bring a charged, a steric, or a specific targeted coating

Many nanomaterials are functionalized on their surface to increase blood circulation,

make them more biocompatible, and for targeted therapy. While surface functionalization has

shown promising applications, functional groups added to the surface can potentially interact

with biological components, alter biological functions, and allow passage of nanomaterials

that would not normally be taken up by certain cells.

- Charged surface treating agent: the interaction with cells membrane

The effect of the surface chemistry of biomaterials on the protein adsorption process

has been a topic of great interest for many years, and much is known in this field. Protein

- 93 -

adsorption to various materials has been widely studied and it has been found that factors such

as electrostatic interactions, hydrophobic interactions, and specific chemical interactions

between the protein and the adsorbent play important roles.

Selective adsorption of proteins on various synthetic adsorbents has been examined

under different conditions (such as solution pH and protein concentration) and for many

proteins the mechanism of selective adsorption has been attributed to electrostatic

interactions.

Figure 29 provides a general recent overview of the major proteins, identified in the

literature, bound to an assortment of nanoparticles of different size, chemical make-up, and

surface properties which compose the nanoparticle protein-corona.

Though some proteins are specific to certain types of nanoparticles, seemingly

dependent on the nanoparticle structure, the proteins that were identified on virtually all

nanoparticles are principally albumin, immunoglobulin G (IgG), fibrinogen, and

apolipoproteins. What is unknown, however, is the affinity of the proteins binding the

nanoparticles. These proteins may dominate the surface of the nanoparticle initially, but then

be displaced by proteins of lower abundance, higher affinity, and slower kinetics. This may

lead to the presence of other nanoparticle-bound proteins at later times, such as other

immunoglobulins, apolipoproteins, and components of the complement system. These

proteins, along with albumin and fibrinogen, are important in the clearance process of the

body.

Figure 29: Summary of various nanoparticles and protein binding (Aggarwal et al., 2009)

Factor B, transferrin, albumin, fibrinogen, IgG, apolipoproteins

Albumin, fibrinogen, apolipoproteins, IgG, �1-antitrypsin, �2-macroglobulin, IgM

Albumin

Fibrinogen, IgG, IgM, apolipoproteins, transthyretin

Fibrinogen, IgG, IgM, apolipoproteins, transthyretin, albumin

Albumin, fibrinogen, IgG, apolipoproteins

Albumin, fibrinogen, IgG, transferrin

IgG, apolipoproteins

Albumin; IgG, IgM, fibrinogen, apolipoproteins

Albumin, IgG, IgM, fibrinogen, C3b, apolipoprotein A-1

Albumin, IgG, fibrinogen, apolipoproteins

Albumin, IgG, fibrinogen, IgM, apolipoproteins, antithrombin III

Albumin, IgG, fibrinogen, apolipoproteins, PLS:6

Albumin, IgG, fibrinogen, IgM, apolipoproteins, PLS:6, U2

Albumin, IgG, fibrinogen, apolipoproteins, serotransferrine, transthyretine

Albumin, fibrinogen, apolipoproteins, C1q

Albumin, IgG, fibrinogen, IgM, apolipoproteins

Albumin, fibrinogen, apolipoproteins

Polystyrene with poloxamer 184, 188, 407Liposomes

Single-walled carbon nanotubes

Solid lipid nanoparticles with Tween 80

Solid lipid nanoparticles with poloxamer 188

Poly(lactic acid) nanoparticles with PEG

Polyhexadecylcyanoacrylate NP

Poly(�-caprolacton) NP

Polycyanoacrylate NP

Iron oxide NP

Various polymer/copolymer composition NP

Poly(D,l-lactic acid) NP

Polystyrene with Rhodamine B


Poly(isobutylcyanoacrylate) with Dextran

Single-and double-walled carbon nanotubesPolybutylcyanoacrylate NP with polysorbate 80

Solid lipid NP with poloxamer or poloxaminecoating

Identified proteinsNanoparticles

Factor B, transferrin, albumin, fibrinogen, IgG, apolipoproteins

Albumin, fibrinogen, apolipoproteins, IgG, �1-antitrypsin, �2-macroglobulin, IgM

Albumin

Fibrinogen, IgG, IgM, apolipoproteins, transthyretin

Fibrinogen, IgG, IgM, apolipoproteins, transthyretin, albumin

Albumin, fibrinogen, IgG, apolipoproteins

Albumin, fibrinogen, IgG, transferrin

IgG, apolipoproteins

Albumin; IgG, IgM, fibrinogen, apolipoproteins

Albumin, IgG, IgM, fibrinogen, C3b, apolipoprotein A-1

Albumin, IgG, fibrinogen, apolipoproteins

Albumin, IgG, fibrinogen, IgM, apolipoproteins, antithrombin III

Albumin, IgG, fibrinogen, apolipoproteins, PLS:6

Albumin, IgG, fibrinogen, IgM, apolipoproteins, PLS:6, U2

Albumin, IgG, fibrinogen, apolipoproteins, serotransferrine, transthyretine

Albumin, fibrinogen, apolipoproteins, C1q

Albumin, IgG, fibrinogen, IgM, apolipoproteins

Albumin, fibrinogen, apolipoproteins

Polystyrene with poloxamer 184, 188, 407Liposomes

Single-walled carbon nanotubes

Solid lipid nanoparticles with Tween 80

Solid lipid nanoparticles with poloxamer 188

Poly(lactic acid) nanoparticles with PEG

Polyhexadecylcyanoacrylate NP

Poly(�-caprolacton) NP

Polycyanoacrylate NP

Iron oxide NP


Poly(D,l-lactic acid) NP

Polystyrene with Rhodamine B


Poly(isobutylcyanoacrylate) with Dextran

Single-and double-walled carbon nanotubesPolybutylcyanoacrylate NP with polysorbate 80

Solid lipid NP with poloxamer or poloxaminecoating

Identified proteinsNanoparticles

- 94 -

Particle adhesion to a cell-surface and engulfment at the adhesion site require specific and

non specific binding interactions (Figure 30).

Among the most effective specific binding interactions are those of surface ligands which

allow the nanoparticle to interact with complementary molecules or receptors on the cell

membrane. These interactions result in receptor-mediated endocytosis. Ligands do not need to

be necessary of biological origin – see for instance gold nanoparticles protein-corona inducing

specific nanoparticles cell uptake (Chithrani et al., 2006). In contrast, they can comprise

chemical moieties, metallic sites, polymers or surface functionalities that promote binding

affinity.

Non specific attractive forces that promote cellular contact and particle uptake result from

intrinsic nanoparticles characteristics such as surface charge, hydrophobicity and roughness.

Among them, surface charge plays an important part in particles interactions with charged

phospholipid head groups or protein domains on cell surfaces. For instance, anionic magnetic

nanoparticles are described to first adsorb electrostatically to the tumour cell membranes

before being internalized within endosomes (Wilhelm et al., 2008). Further, Bose et al., 2004

have reported the binding between apoptotic leukemic T cells and cationic liposomes (100-

115 nm). Results showed that the binding of liposomes is mediated through an electrostatic

interaction between the positively charged liposome and the translocated anionic

phosphatidylserine in the plasma membrane.

Figure 30: For particle uptake to occur, specific (ligand–receptor) and nonspecific (for example

hydrophobic, coulombic). Binding interactions must decrease the free energy at the contact site to overcome resistive forces. The blue ellipses represent clathrin, an example of an endocytic component

that engages in energy-dependent uptake of particles. The red spheres represent nanoparticles with attached ligands (yellow dots). These ligands bind to the Y-shaped membrane receptors.

- 95 -

- Targeting the nanoparticles surface: which option?

- The passive surface targeting: EPR effect

First demonstrated by Maeda et al., in 1989, the EPR phenomenon is based on two

factors:

- The capillary endothelium in malignant tissues is more disorderly and thus more

permeable towards macromolecules than the capillary endothelium in normal tissues.

Tumours grown subcutaneously have shown to exhibit a characteristic pore cutoff size

ranging from 200 nm to 1.2 µm, the majority ranging between 380 and 780 nm (Hobbs et al.,

1998). This allows extravasation of circulating polymeric nanoparticles within the tumour

interstitium. The increased permeability of the blood vessels in tumours is characteristic of

rapid and defective angiogenesis (formation of new blood vessels from existing ones). The

hyperpermeable nature of the tumour microcirculation is well described. Numerous

morphological studies have demonstrated the existence of interendothelial junctions,

transendothelial channels, fenestrations, and vesicular vacuolar organelles in tumour vessels.

- The lack of tumour lymphatic drainage in the tumour bed results in drug

accumulation.

Most polymer nanoparticles display the EPR effect. If a chemotherapeutic agent is

coupled to a suitable polymer or other molecular carrier via a degradable linker, then such

carriers have the potential of increasing the concentration of the chemotherapeutic agent

within the tumour tissue. As a result of these characteristics, concentration of polymer-drug

conjugates in tumour tissue can reach levels 10 to 100 times higher than that resulting from

the administration of the free drug (Maeda et al., 2009).

Most importantly, EPR effect can be observed in almost all human cancers with the

exception of hypovascular tumours such as prostate cancer or pancreatic cancer. As clinical

examples, Doxil, a liposomal formulation of doxorubicin injected via the hepatic artery,

accumulated selectively in hepatocellular carcinoma. Mechanisms and factors involved in the

EPR effect, as well as the uniqueness of nanoscale drugs for tumour targeting through EPR

effect, are discussed in detail in many reviews (Greish et al., 2007; Maeda et al., 2001 and

2009). Meada et al., 2009 described the factors facilitating EPR effect such as bradykinin,

nitric oxide (NO), VEGF, prostaglandins (PG) and matrix metalloproteinases (MMP). EPR

- 96 -

effect can be further augmented by elevating the systemic blood pressure artificially by

infusing angiotensin-II, and also by prostaglandin (PG) I2 agonist. Consequently, the delivery

of macromolecular drugs is facilitated significantly even to metastatic tumour which has low

vascular density (Maeda et al., 2009).

The most commonly used strategy, to achieve “sterically stabilized” nanoparticles is to

conjugate polyethylene glycol (PEG) polymers onto the surface of the nanoparticles. A great

deal of work has been devoted to developing the so-called “stealth�” particles which are

invisible to macrophages, hence increasing nanoparticles blood circulation which is a pre-

requisite for EPR effect. PEG is a relatively inert hydrophilic polymer which provides good

steric hindrance for preventing proteins binding. Li et al., 2008 have demonstrated that

PEGylation reduces the rate of mononuclear phagocyte system (MPS) uptake and increases

circulation half time for various types of nanoparticles, including liposomes, polymer-based

nanoparticles and hybrid nanoparticles. The area under curve (AUC) of the blood pK profile

of the drug irinotecan hydrochloride (CPT-11) formulated in the PEGylated liposome was 6-

folds higher than that of non-PEGylated formulation and 36-folds higher than that of the free

drug. Akiyama and his colleagues grafted various amounts of PEG onto the surface of gold

nanorods and investigated the effects of grafting amount and injection dose on the

biodistribution in the tumour-bearing mice (Colon-26) after i.v injection. Higher PEG grafting

amount were advantageous for reticuloendothelial system (RES) avoidance and for

suppression of aggregation of the gold nanorods in the circulation. A PEG, Au molar ratio of

1.5 was sufficient to confer prolonged circulation of gold nanorods in the blood and to show

an EPR effect (Akiyama et al., 2009).

- Active surface targeting

To target cell or tissue structures, two general approaches are developed which offer

improvement of agent-biological interactions.

Healthy and tumour tissues present differential genotypes with dynamic phenotypes

and these differences can be exploited for targeting key biological features present in one

system and absent in the corresponding one. Growth factor or vitamin interactions with cancer

cells represent a commonly used targeting strategy, as cancer cells often overexpress the

receptors for nutrition to maintain their fast-growing metabolism. EGF has been shown to

block and reduce tumour expression of the EGF receptor, which is overexpressed in a variety

- 97 -

of tumour cells such as breast and tongue cancers. Additionally, based on the same idea, the

vitamin folic acid (folate) has also been used for cancer targeting because folate receptors are

frequently overexpressed in a range of tumour cells including ovarian, endometrial and kidney

cancers. Transferrin interacts with transferrin receptors, which are overexpressed on a variety

of tumour cells (including pancreatic, colon, lung, and bladder cancers) owing to increased

metabolic rates.

The second modality is determined by the intention to target particular molecular

features of specific cancers and disease stage. There is clinical evidence that trastuzumab, a

monoclonal antibody targeting the human EGFR HER, a two tyrosine kinase receptor, is an

important component of first-line treatment of patients with HER2-positive metastatic breast

cancer (Nielsen et al., 2009).

Antibodies were the first macromolecular ligands used for targeted delivery. The use

of mAb became widespread after the discovery of hybridoma technology. Due to their

inherent immunogenicity, murine mAb were not suitable for clinical applications.

Engineering antibody technologies led to the development of chimeric humanized and fully

humanized antibodies. More recently, methods have been developed to produce human

immunoglobulins from transgenic mice. Combinatorial phage display libraries have also

emerged as a powerful tool to select novel protein ligands. Research using antibodies for

targeting applications has led to a better understanding of critical factors affecting targeting.

Peptides and antibody fragments have been developed to overcome some of the shortcomings

of antibodies, and several examples of these ligands are now under clinical development

(Alexis et al., 2008).

It is now well accepted that the binding affinity, stability, and the size of the ligand

play a critical role for successful targeting.

Peptides are small, synthetic molecules that can be manufactured in large quantities

with excellent quality control. Peptides are more stable than antibodies and unlikely to be

immunogenic.

Nucleic acid aptamers are single stranded DNA, RNA or unnatural oligonucleotides

which fold into unique structures capable of binding to specific targets with high affinity and

specificity (Alexis et al., 2008).

Small molecules such as multitargeted tyrosine kinase inhibitors represent

simultaneous interruption of tyrosine kinases and serine/threonine kinases pathways. They

serve to block angiogenesis and tumour cell proliferation.

- 98 -

A targeting ligand conjugated to the surface of nanoparticles can recognize and bind

receptors, membrane proteins, surface antigens expressed on the target cell surface, which

later triggers endocytosis, resulting in an increased level of intracellular delivery. This

approach is particularly useful for delivering molecules with low membrane permeability,

such as “druggable” DNA, oligonucleotide, and siRNA. Several active targeting strategies

(antibodies, carbohydrates, peptides, and small molecule ligands) have been developed and

showed promising results (Li et al., 2008).

- Both passive and active targeting could be the best compromise for efficient

nanoparticles bioavailability

As a summary, passive tissue targeting may be achieved by extravasation of

nanoparticles through increasing permeability of the tumour vasculature and ineffective

lymphatic drainage, using proper nanoparticle surface functionalization, conferring the so

called “stealth�” effect. Further, active cellular targeting may be achieved by functionalizing

the nanoparticles surface with ligands that promote cell-specific recognition and binding.

Figure 31 summarizes the two approaches, which may be viewed as complementary

approaches: targeted nanoparticles concentrate in the tumour interstitium through the EPR

effect, exactly as do nanoparticles designed for passive tissue targeting, but once there,

nanoparticles are actively taken up by cancer cells after binding to their target antigens on the

surface of the cancer cells (Alexis et al., 2008).

Interestingly, Couvreur et al., 2006 have shown, using liposomes, that decorating the

surface of the nanocarriers with antibodies directed against tumour-associated antigens needs

to be balanced between a sufficient number of antibody molecules per nanocarriers surface to

achieve efficient binding and recognition on one hand, and not too many antibodies to avoid

complement activation and to keep the ability of the immunovesicles to escape from the

recognition by the MPS on the other hand. An optimal coating of 10-30 antibody molecules

per nanocarriers seems to allow the combination of an efficient delivery with a limited uptake

by the MPS.

- 99 -

Figure 31: Active and passive targeting of nanoparticles (Peer et al., 2007)

3.3. Toxicological issues: probably the most important concern to address when designing a nanomaterial

An understanding of the hazardous properties of nanomaterials is essential if they are

to come into contact with humans. The nanoparticle/biological interface which arises when

nanoparticles come into contact with human, embraces particle protein corona formation,

particle membrane wrapping, particle trafficking at the subcellular level, which are of most

concern when addressing the nanomaterial safety issue.

Nanoparticle protein corona may induce changes in protein structure and function and

can lead to potential mechanism of injury that could contribute to disease pathogenesis.

Figure 32 illustrates the potential effects on protein structure and function following

interaction with nanoparticles. Particles composition at the nanoscale level confers to the

nanoparticles their performance characteristics (electronic, magnetic properties among

others). However, such characteristics may be responsible for protein structural or functional

changes. It is possible that electron confinement or the formation of electron hole pairs at the

material surface could lead to cleavage of structural bonds or covalent cross-linking of protein

- 100 -

SH domains. The interaction between human adult hemoglobin (Hb) and bare cadmium

sulfide (CdS) quantum dots (QD) has been investigated by fluorescence, synchronous

fluorescence, circular dichroism (CD) and Raman spectroscopic techniques under

physiological pH 7.4. CdS QD dramatically alter the conformation of Hb, quenching the

intrinsic fluorescence of Hb and decreasing the α-helix content of the secondary structure

from 72.5% to 60.8%. Raman spectra results indicate that the sulfur atoms of the cysteine

residues form direct chemical bonds on the surface of the CdS QD (Shen et al., 2007). Along

similar line, when a protein containing cryptic epitopes is denatured on a particle surface, the

exposure of new antigenic sites may initiate an immune response, which, if launched against

self-protein, could promote autoimmune disease. Here, more than the nanoparticle size or

nanoparticle surface area, the nanoparticle reactive surface area constitutes a more accurate

measure of particle potential toxicity. While, in biological environment, the nanoparticles

agglomeration behaviour takes a role in defining the reactive surface area. Indeed,

nanoparticle surface functionalization may influence nanoparticles agglomeration behaviour

in biological media – by promoting particles dispersion or, on the contrary, leading to

nanoparticles agglomeration – or directly inflicts the reactive surface area.

Figure 32: The corona constitutes a primary nano–bio interface that determines the fate of the nanoparticle and can cause deleterious effects on the interactive proteins. Potential changes in protein

structure and function as a result of interacting with the nanoparticle surface can lead to potential molecular mechanisms of injury that could contribute to disease pathogenesis. The coloured symbols represent various types of proteins, including charged, lipophilic, conformationally flexible proteins,

catalytic enzymes with sensitive thiol groups, and proteins that crowd together or interact to form fibrils (Nel et al., 2009).

- 101 -

Particle adhesion to a cell-surface bilayer and particle engulfment at the adhesion site

is the result of promoting forces (such as specific binding induced by ligand-receptor

interaction, non specific binding due to particles surface characteristics, optimal particle size

and shape,...) and resistive forces (such as stretching and elasticity of cell membrane, receptor

diffusion to adhesive front,...). Nanoparticles surface charge plays an important part in

particles interactions with charged phospholipid head groups or protein domains on cell

surfaces. Nanoparticles with cationic surface charge exert generally stronger effect than their

anionic counterparts. However, if the nanoparticles cationic density is not controlled, the

interactions may compromise the cell membrane integrity. Nanoparticles shape is also an

important factor for nanoparticles cell uptake; studies of pulmonary exposure of pure single-

walled carbon nanotubes (SWCNT) have shown that SWCNT are poorly phagocytized by

alveolar macrophages; instead, they migrate to the interstitial space in the alveolar wall,

where, after close contact with fibroblasts, they directly stimulate cellular proliferation and

collagen production.

Nanoparticle trafficking at the subcellular level may exert important effects on

organelles. Particularly noteworthy is the impact on lysosomal function. The lysosomal proton

pump, which is responsible for acidification, is key to understanding the proton sponge

hypothesis, which posits that unsaturated amines on nanoparticles surface are capable of

sequestering protons, keeping the pump going and leading to the retention of one Cl- anion

and one water molecule for each proton that enters the lysosome. Ultimately, this process

causes lysosomal swelling and rupture. Spillage of the lysosomal content may lead to toxicity.

Lysosomes are also involved in nanoparticles dissolution, as illustrated by the toxicity of ZnO

or CdSe.

Hence, when designing nanomaterials, the optimal design process appears one of

multiple compromises. Above all, potentially competing principles (aggregation, dispersal,

transport, bioavailability) must be analysed in detail to formulate the best design strategy for

safe but efficient use of the nanoparticles in their intended purpose. Interestingly, surface

coating is perceived as an attractive approach to improve nanoparticles safety by playing

different roles such as preventing bioreactivity and dissolution. Nanoparticles surface

functionalization may also modulate nanoparticles biodistribution and/or nanoparticles

cellular trafficking and may be considered for a safe use of nanoparticles in biological media.

However, many coatings are recognized as being environmentally labile or degradable, hence

an initially non-toxic material may become hazardous after shedding its coat.

- 102 -

4. Summary of PhD study: participation to the development of two types of

nanoparticles intended to treat cancer with different rational for design

The present PhD study focuses on the 2 major advanced researches developed by

Nanobiotix: the nanoXrayTM technology platform and the nanoPDT technology platform.

Products issued from those platforms aim to create and develop effective nanomaterials to

treat cancer, using external energy sources to carry the “on” / “off” activation.

However, the energy sources used to “activate” the products are notably different

(Figure 33). Furthermore, the route of administration is also different; meaning that the

rational for design of each nanoparticle is fully different. Further, the platforms are dedicated

to radically different therapeutic approaches which share the endpoint of delivering loco-

regional control of diseases.

NanoXrayTM platform creates and develops nanomaterials which are designed to

interact with X-ray radiations to subsequently enhance locally the effect of radiotherapy.

NanoXray™ products are inert and electron-dense particles which generate electrons with

kinetic energy that will be released into the medium and will generate free radicals, following

X-ray absorption. The nanoparticles have an electron density much higher than water, so they

have a far more powerful cancer killing effect than standard radiotherapy, and have fewer

safety issues than currently available cancer treatments. NBTXR3 is the lead product of

nanoXrayTM platform, synthesized to enter within the tumour cell by direct injection, without

any specific target or interaction. NBTXR3 is a suspension of inert crystalline nanoparticles

(±70 nm) of coated hafnium oxide (HfO2), in water for injection. The NBTXR3 oxide

nanoparticles have a simple composition. The hafnium oxide core represents the active

component, but only when its electrons are excited by the application of an external beam of

X-ray. This core is coated by a layer which ensures stability in fluids and improves

biocompatibility.

Moving to longer wavelength throughout the electromagnetic spectrum, the secondary

electromagnetic energy source used by Nanobiotix lies in the near-infrared region. Such

wavelength of light is able to penetrate biological tissues and may be exploited for clinical

purpose. NanoPDT platform has developed nanoparticles that are designed to interact with

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laser sources. Existing organic photosensitizers have currently demonstrated interesting

efficacy and some of them have received market approval. But their difficulty to perform

breakthrough in the clinic relies on their poor pK and their lack of selectivity toward targeted

tissues. NanoPDT product (or silica-based nanoparticle) is intended to be administered by

intra-venous injection. NanoPDT product consists in a silica-based nanoparticle which

successfully encapsulates the photosensitizer to readily improve its pK while preserving its

ability to generate reactive oxygen species (ROS) to the surrounding media upon laser light

excitation.

Figure 33 : NanoPDT and nanoXrayTM spectrum of activation

NanoXrayTM oxide nanoparticles activated with typical radiotherapy treatments or

nanoPDT nanocarriers for PDT have clearly for ambition to enlarge the therapeutic window.

PhD studies take part to 3 interconnected research axes which are key when establishing the

rational for development of the therapeutic products.

nanoXraynanoXraynanoPDTnanoPDT nanoXraynanoXraynanoPDTnanoPDT

- 104 -

In vitro efficacy and toxicity studies

In vitro approach is of particular relevance to assess the nanoparticles toxicity, the

nanoparticles efficacy, the nanoparticles subcellular localization effect on efficacy, the type of

cellular death, but also to screen different tumour cell types as well as to study the effect of

nanoparticles on normal cells. Indeed, many questions may be underpinned in in vitro studies

when considering the nanoparticle as the ‘active product’ to enlarge the therapeutic window:

- Are the nanoparticles toxics without activation - for both tumour and healthy cell

lines?

- Do nanoparticles enhance tumour cells destruction upon activation by external energy

sources - according to schedules and protocols?

- Which sort of cell death is induced by the nanoparticles?

- What are the effects of nanoparticles subcellular localization on efficacy?

- Do nanoparticles induce the same efficacy / toxicity according to tumour biologic

specificities: their origin (epithelium and mesenchymal origin, different

compartmental structure), human healthy versus malignant tumour cells,

radiosensitivity characteristics (radio-sensitive or radio-resistant cancer types)?

Mechanism of action: nanoparticles trafficking at cellular level

Nanoparticle trafficking at cellular level is a key understanding to yield foundations to

establish the schedule of nanoparticles administration in in vivo models and the modality of

energy source delivery. In vitro studies of nanoparticles uptake, localization and clearance are

of particular relevance to precise nanoparticles trafficking at the cellular level. Many research

axes need to be explored:

- What is the nanoparticles uptake mechanism?

- What is the kinetic of nanoparticles uptake?

- Where are the nanoparticles localized within cells?

- How many nanoparticles are internalized per cells?

- Are nanoparticles exocytosed or are they trapped within organelles and ultimately

degraded within the cells?

- 105 -

In vivo performance and safety

In vivo studies encompass the nanoparticles biodistribution, the nanoparticles tumour

accumulation and dispersion, the nanoparticle toxicity. Tumours are specific “organs”,

different from the organ/tissue from which they arose. These 2 genetic entities coexist, and

have molecular structure, metabolic and growth behaviors particular to the patient and closely

related to the tissue type and cancer disease stage. Exploration of biologic specificity of

different cell/tissue/organ is needed. In vivo studies must be sustained by in vitro studies to

bring safe and efficient products in the clinic. Research axes are multiple and some of them

are highlighted in the following questions:

- What is the ideal nanoparticle (according to its size, composition, shape and surface

coating) in order to optimize biodistribution and/or cellular uptake and/or localization

at the subcellular level?

- What is the ideal nanoparticle (according to its size, composition, shape and surface

coating) for optimized biocompatibility?

- Are nanoparticles stable in biological media?

Ultimately, the aim of all those studies is to show the benefice of nanoparticles over

the current therapeutic approaches. For nanoXrayTM applications, the reference control is the

radiotherapy alone whereas for nanoPDT applications, the reference control is the free drug.

� Presentation of the PhD work objectives: study of interactions between

nanoparticles and cancer cells

Nanobiotix is working on oncology field and therapeutic window enlargement is the

main concern in the clinic. Nanobiotix has explored two different pathways: inert therapeutic

nanoparticles activated by radiotherapy for local radiation enhancement and silica-based

nanoparticles for improved drug delivery for PDT. Specifically, study of interactions between

nanoparticles and cancer cells was explored.

The following Figure 34 summarizes the work undertaken during this PhD project.

- 106 -

Chemistry

CLEARANCE

Localisation

Total amount of NP internalisation per cell

UPTAKE

In vitro

In vivo

Cellular death by ROS liberation

Cytotoxicity of NP

Stability of NP in FCS

NP efficacy > product of reference or therapy

alone

Clinical studies Biodistribution

Efficacy

Impact ofsize/shape/coating-

charge

Comparison of NP cellviability in different cells

lines

Get rational for clinicalstudies

Chemistry

CLEARANCE

Localisation

Total amount of NP internalisation per cell

UPTAKE

In vitro

In vivo

Cellular death by ROS liberation

Cytotoxicity of NP

Stability of NP in FCS

NP efficacy > product of reference or therapy

alone

Clinical studies Biodistribution

Efficacy

Impact ofsize/shape/coating-

charge

Comparison of NP cellviability in different cells

lines

Get rational for clinicalstudies

Figure 34: Study of interactions between nanoparticles and cancer cells

NBTXR3 nanoparticles interaction with human tumour cells – binding to membranes

efficiency, intracellular uptake, nanoparticles localization and change over time – the effect of

nanoparticles localization on efficacy, the nanoparticles dose effect on efficacy, were all the

aspects covered by the present study, with the ultimate goal to yield foundation for schedule

of NBTXR3 nanoparticles administration in in vivo models and the modality of ionizing

radiation delivery.

NanoPDT interaction with human tumour cells – intracellular uptake, amount of

nanoparticles per cell and kinetic of clearance – were studied to define the optimum schedule

for nanoparticles activation. NanoPDT ROS generation quantification and ROS subcellular

localization were also covered by the present study. Comparison of nanoPDT biodistribution

in different in vivo models was underpinned to better understand the role of cell type,

engraftment site and stroma contribution.

- 107 -

NanoXray™ therapeutic products allow dramatic

increase of X-ray dose on tumour without changing

dose applied to healthy tissues. NanoXray™

increases local destruction of the tumour without

modifying healthy tissues

- 108 -

1. Nanoparticles activated by X-ray: NBTXR3 mechanism of action

As it is well known, ionizing radiation interacts with atoms or molecules in the cells,

particularly with water, to produce free radicals which are responsible for non specific cellular

damages, leading to subsequent cell death. As a result of the interaction with a photon, the

water molecule will ionize, producing a fast e- , a photon of reduced energy, and an ionized

water molecule: H2O � H2O+ + e-. The ionized water molecule will de-excite either by

producing Auger e- or X-ray photon and free radicals. Fast electrons that will be released into

the medium will generate free radicals or heat. These electrons with kinetic energy are

responsible for the largest effect in radiotherapy.

Hafnium oxide core of nanoXrayTM products will interact with a photon or an electron

in exactly the same way. However, the probability of absorption (of a photon or an electron)

is proportional to the density and the atomic number (Z) of the absorbing material. Water has

d = 1, Z = 8, whereas nanoparticles have d > 8, Z > 60. Hence, hafnium oxide nanoparticles

will generate the same type of effect as water molecules, but by several orders of magnitude

higher.

Primary mode of action of NBTXR3 product

The mechanism of action of nanoparticles activated by X-rays is based on a physical

and energetic effect and not on pharmacological, immunological or metabolic effect. The

physical and energetic mechanism of action of such a product is based on electron ejection

from HfO2, which generates free radicals after X-ray energy exposure.

The mode of action of NBTXR3 is described as follows:

Step 1- On/off activation: Energy absorption, electrons emission, and free

radicals generation

NBTXR3 works according to an “on-off” activity status:

• When the nanoparticles are not activated, they do not have any effect because they are

inert.

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• Under external beam X-ray activation as customarily used in radiation oncology,

- X-rays are absorbed by HfO2 (NBTXR3) exactly as ionizing radiations are

absorbed by water molecules. Meaning an X-ray photon will interact losing a

part of its energy with an electron from the atom and will create i) a hole , ii)

an electron with kinetic energy and iii) the incident photon will have lost a part

of its energy and could participate to subsequent interactions (Figure 35).

- The electron created will travel in the cellular medium and will lose its energy

by interacting several time with water molecules creating free radicals (mainly

responsible for cellular damages). See step 2 for cellular damages induced by

such free radicals.

- The hole created in the atom will come back to its initial state by taking an

electron from the water molecule in the medium.

1) Xray photon

With energy hν(X) interacting with water

hν’(X)Principal radiotherapy mechanism of action is based on photon/electroninteraction

e- with kinetic

energy

R4) Free radical generated after

electron/water interaction will be

responsible for non-specific cellular

damage , leading to subsequent cell

death

hν(X)

2) Creation of an electron (that takes part of the incident energy photon) and a secondary photon that will have lower energy than incident photon

3) Electron will travel in water medium within the cell and outside cell and will lose its energy by interaction mainly with water creating free radical

This secondary photon can interact with another molecule of water (step 1)

R

R

R

Electron pathway

A

1) Xray photon



e- with kinetic

energy





death

hν(X)




R

R

R

Electron pathway

A

1) Xray photon



e- with kinetic

energy





death

hν(X)




R

R

R

Electron pathway

A

- 110 -

Figure 35: Principal radiotherapy mechanism of action of A/H2O ; B/NBTXR3 The nanoparticles do not react directly with any biological recipient cell and tissue.

The X-ray irradiation can be applied several times on the same nanoparticle. When the X-ray

irradiation is off, NBTXR3 returns to its inactive state.

Step 2- Cellular damage

The free oxygen radicals generated by electrons ejected from the HfO2 (NBTXR3) and water

are quite reactive as they are attempting to form covalent bonds with another species. They

can lead to non-specific damages to subcellular organelles and biomolecules around the

location of nanoparticles or along the electron pathway that will create free radicals.

Step 3- Subsequent actions on cells

The mode of action of NBTXR3 is mainly explained by the effect of free oxygen radicals

leading to cytotoxic effects through non-specific damages to tumour cells. Resultant cellular

1) Xray photon

With energy hν(X) interactingwith nanoparticle

hν’(X)Principal radiotherapynanoXray mechanismof action is based on photon/electroninteraction

e- with kinetic

energy





death

hν(X)

2) Creation of an electron (that takes part of the incident energyphoton) and a secondary photon thatwill have lower energythan incident photon

3) Electron will travel in water medium withinthe cell and outside celland will lose its energyby interaction mainlywith water creating freeradical

This secondary photon can interact withanother molecule ofwater or anotherparticle (step 1)

R

R

R

Electron pathway

B

1) Xray photon



e- with kinetic

energy





death

hν(X)




R

R

R

Electron pathway

1) Xray photon



e- with kinetic

energy





death

hν(X)




R

R

R

Electron pathway

1) Xray photon



e- with kinetic

energy





death

hν(X)




R

R

R

Electron pathway

B

- 111 -

destruction is induced by the usual effect of free radicals as for radiotherapy, but enhanced

because of the nanoparticles.

All these physical mechanisms mentioned above, can explain the effect of NBTXR3 on

tumour cells and support the use of such a therapy in cancer treatment. This mode of action

actually mimics the ionizing radiation mode of action on biological systems (Perez et al.,

2004; Kufe et al., 2006 and Zhou et al., 2004) (Figure 36).

Figure 36: Cell radiation responses (Gudkov et al., 2003)

2. Metallic nanoparticles activated by ionizing radiation to treat cancer

Gold nanoparticles literature survey

The following literature tends to expose the rational for development of gold

nanoparticles activated by ionizing radiations to treat cancers. Gold nanoparticles present a

high Z (=79) which is expected, providing intra-tumour gold nanoparticles localization, to

enhance the dose delivered to a tumour during photon-based radiation therapy, resulting in a

differentiate efficacy between tumour and surrounding tissues. Cho et al., 2005 have

performed Monte Carlo calculation to estimate the dose enhancement of gold nanoparticles

uniformly distributed throughout a tumour, provided some insight into the amount of

achievable dose enhancement of gold nanoparticles activated by typical radiation treatment,

with a significant higher dose enhancement estimated in the kilovoltage energy range.

- 112 -

Despite the establishment of in vitro and in vivo efficacy using gold nanoparticles

activated by ionizing radiations – supporting the theoretical Monte Carlo calculation –gold

nanoparticles safety studies are scarce and disparate and it seems difficult to get a clear view

of their potential toxicity in the clinic.

In vitro efficacy and mechanism of action

In their article, Kong et al., 2008 report an in vitro performance study of home-made

10.8 nm gold nanoparticles activated by ionizing radiations to treat cancer cells. They put

forward (i) the importance of gold nanoparticles localization at the cellular level, (ii) the role

of the ionizing radiation energy source and (iii) the differential effect of radiation sensitivity

observed in cancer cells versus nonmalignant cells.

The authors have used different surface treating agent, thioglucose (GLU) and 2-

Aminoethanethiol (AET) to functionalize gold nanoparticles. Following incubation of

functionalized gold nanoparticles with MCF7 breast cancer cells, gold nanoparticles

functionalized with thioglucose were mostly found distributed in the cytoplasm of cells. On

contrast, gold nanoparticles functionalized with AET were mostly found bound to the cell

membranes. The authors have performed in vitro MTT assays using the same level of gold

nanoparticles per cell for either AET-gold nanoparticles or GLU-gold nanoparticles. Cells

either untreated or treated with functionalized gold nanoparticles were irradiated with 200

KVp X-rays with a dose of 10 Gy. GLU-gold nanoparticles and AET-gold nanoparticles were

found to induce about 63.5% and 31.7% increase in radiation cytotoxicity, respectively, when

compared to irradiation alone. Additional clonogenic survival assays were consistent with

previous results. The authors stated that this phenomenon indicates that the location of gold

nanoparticles in the cells is an important factor in the increase of radiation cytotoxicity

induced by gold nanoparticles. Interestingly, under similar conditions, the authors found that

gold nanoparticles increase the radiation sensitivity of MCF7 cancer cells but not that of

nonmalignant cells MCF-10A. However, no significant dose enhancement was observed

when compared to control, using gold nanoparticles activated by high energy radiation source

(60Co, 137Cs or �-rays).

Rahman et al., 2009 present an in vitro performance study of 1.9 nm gold

nanoparticles activated by two different energy sources using bovine aortic endothelial cells.

The article put forward, the importance of (i) gold nanoparticles concentration at cellular level

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and (ii) the role of the energy source, for an enhancement of radiation effect. Interestingly, a

dose enhancement effect was observed for cells treated with gold nanoparticles and irradiated

using high energy electron beams. Cytotoxicity was observed with gold nanoparticles

concentration of 0.25 mM and beyond. Toxicity issue will be discussed specifically in later

paragraph, when addressing the safety of gold nanoparticles.

Two energy sources were tested. Studies were done with kilovoltage superficial

radiation therapy-type X-rays (80 kV and 150 kV X-rays energy at various dose 0, 1, 2, 3, 4

and 5 Gy) and megavoltage electron beams (6 MeV and 12 MeV). Cells in vitro were treated

with increased concentration of 1.9 nm gold nanoparticles from Nanoprobes (from 0.25 mM

up to 1 mM). After continuous exposure of the cells to the gold nanoparticles for 24 hours,

gold nanoparticles, for each concentration tested, were found internalized within the

cytoplasm of cells as clusters. Dose enhancement, in cells irradiated with superficial X-rays,

was concentration-dependent and energy-dependant. Similar to the superficial X-ray, the

results with cells irradiated with electron beams (both 6 MeV and 12 MeV) under identical

radiation doses showed that cells containing gold nanoparticles presented a lower survival

percentage with increasing concentration of gold nanoparticles, highlighted the ability of gold

nanoparticles to enhance the effect of radiation even under high energy electron sources.

In vivo efficacy and mechanism of action

Hainfeld et al., 2004 have established the first proof of tumours eradication, when

using gold nanoparticles (1.9 nm from nanoprobes) activated by X-rays following i.v

injection.

Syngeneic mouse mammary EMT-6 tumours were grown subcutaneously in the legs

of mice. After i.v injection of gold nanoparticles, the tumour region was irradiated with 250

kVp X-rays. A dose of 30 Gy was delivered in a single fraction. Irradiation was performed 2

min following i.v injection. Animal receiving either no radiation or gold without radiation

died within 2 weeks. Irradiation alone slowed tumour growth and resulted in 20 % long-term

remissions. Animals receiving injection of 1.35 or 2.7 g Au/kg body weight before irradiation

showed 50 % and 86 % long term remission. pK, showed an early and rapid rise followed by

a slower clearance rate. Gold in tumour peaked at 7.0 ± 1.6 min and fell to one-half of its peak

value at 41.2 ± 19.5 min. gold in muscle peaked at 5.3 ± 0.6 min and fell to one-half at 24.2 ±

2.6 min. The gold nanoparticles cleared nearly twice as fast from normal muscle as from

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tumour. The preliminary toxicity testing on mice receiving 2.7 g Au/kg lived for more than 1

year without overt clinical signs. Analysis of blood from mice having given 0.8 g Au/kg, 2

weeks after injection showed haematocrits and enzymes within normal range. As perspective

from their preliminary results the authors proposed to explore further the potential of gold

nanoparticles to enhance the effect of radiotherapy with the following approaches: use of

megavoltage sources, direct intratumoural injection, second-generation gold agent with

targeting molecule, fractionated radiotherapy. This last approach is of particular relevance as

fractionated radiotherapy represents the standard of care of most clinical protocols. In the

present study, irradiation is performed 2 min post i.v injection. Obviously, gold nanoparticles,

used in the present article, present rapid tumour clearance which appears as a key issue in the

context of fractionated radiotherapy. Furthermore, considerable dose (30 Gy) was delivered to

the tumours. Hence, those gold nanoparticles may be objected as potential product for

development in the clinic.

Chang et al., 2008 present an in vivo performance study, using home-made 13 nm gold

nanoparticles with sodium citrate acting both as reducing agent and as surface complexing

agent, in combination with 6 MeV electron beam to treat tumour. The article highlights the

potential of 13 nm gold nanoparticles to significantly retard tumour growth and to prolong

survival compared to the radiation alone.

Syngeneic mouse mammary melanoma B16F10 cells were grown subcutaneously in

the legs of mice. Gold nanoparticles, 1 g/kg of mice body weight were intravenously injected.

Approximately 24 hours post-gold nanoparticles injection, the tumour region was irradiated

with 6 MeV electron beam. A single dose of 25 Gy was delivered to the tumour. The results

revealed that tumour growth was both retarded in mice receiving either radiation alone or

receiving gold nanoparticles followed by radiation. But more importantly, tumour volume in

the combination therapy group was significantly smaller compared with that in radiation alone

group, whereas administration of gold nanoparticles alone did not exert any antitumour effect

on tumour-bearing mice. Biodistribution of gold nanoparticles was performed 24 hours post-

i.v injection. A notable accumulation of gold nanoparticles inside the tumour tissues was

detected. The tumour-to-tumour surrounding muscle gold ratio was 6.4:1. Nevertheless,

higher concentrations of gold nanoparticles were also found in the spleen and the liver, which

indicated that the gold nanoparticles were also uptaken by the reticuloendothelial system.

Apoptosis in tumours with gold nanoparticles without and with radiotherapy was observed in

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TUNEL-stained cryosections and compared to control (no particles) without and with

radiotherapy. Noticeably, the number of apoptotic cells detected was significantly higher in

the gold nanoparticles and radiation combination group than in the radiation alone group. For

a deeper understanding of the mechanism of action, the authors have performed in vitro

visualization of gold nanoparticles at the cellular level. Eighteen (18) hours post incubation,

gold nanoparticles were found inside the B16F10 cells, localized in the ER and Golgi

apparatuses. The authors mentioned that continuous ER stress results in apoptotic cell death;

therefore, the accumulation of gold nanoparticles in ER and Golgi may also contribute to the

increase of the apoptotic potential of cells post irradiation.

The outcome of those two preclinical in vivo studies is summarized in the following:

Authors GNP size

Tumour to normal tissue

ratio peak

Clearance (half peak)

Time before irradiation

after i.v

Irradiation dose

Hainfeld 1.9 nm 3.5 / 1

at 5 min post i.v tumour: 41 min normal : 24 min

2 min 30 Gy

250 kVp X-ray

Chang 13 nm 6.4 / 1

at 24 hours post i.v

/ 24 hours 25 Gy

6 MeV e-

Gold nanoparticles with increased nanoparticles size seem more interesting when

considering gold nanoparticles retention within tumour, particularly in the perspective of

fractionated radiation protocol if multi-nanoparticles injections are to be excluded. However,

very high doses are required (or at least presented) in the current studies, which seem quite

unrealistic for the clinic, still in the context of fractionated radiotherapy.

Gold nanoparticles trafficking at cellular level.

Chithrani et al. (2006, 2007 and 2009) have deeply explored the effects of gold

nanoparticles size, shape and surface coating on intracellular uptake, transport and subcellular

nanoparticles localization. HeLa, MCF7, STO (fibroblast) and SNB19 (brain) cell lines were

alternatively used for their studies. Spherical gold nanoparticles were synthesized with size of

14, 50 and 74 nm using citric acid as both reducing and complexing agent. Uncoated gold

nanoparticles were stabilized in aqueous media by citrate ions, conferring a negative surface

- 116 -

charge to the nanoparticles. Bovine Serum Albumin (BSA) or transferrin proteins were also

used as surface treating agent.

Gold nanoparticles group in small vesicles of approximately 500 nm in diameter in the

cells cytoplasm, with no nanoparticles found alone in the cytoplasm. Within the vesicles, the

nanoparticles appear to be monodisperse.

Authors suggest that uptake of uncoated gold nanoparticles is mediated by non

specific adsorption of serum proteins onto the gold surface. This is likely since citric acid

stabilizers are weakly bound to the surface of gold nanoparticles and could be desorbed from

the metal surface by proteins. These proteins induce the nanoparticles to enter cells via the

mechanism of receptor-mediated endocytosis – indeed, in vitro nanoparticles cells uptake

studies performed at 37°C versus 4°C support their hypothesis. Additionally, their observation

of an uptake saturation curve as a function of gold nanoparticles concentration is commonly

observed for receptor-mediated endocytosis. Whatever gold nanoparticles size, gold

nanoparticles uptake reaches saturation following 6 hours of incubation with cells. Gold

nanoparticles, either pre-coated with BSA or transferrin proteins, show similar cells uptake,

but to a lesser extend when compared to uncoated gold nanoparticles. The authors state that

the surface of the initial uncoated gold nanoparticles (citrate-stabilized nanoparticles) contains

a variety of serum proteins and that their diversities may allow entrance into cells via multiple

receptors.

Specific studies with gold nanoparticles pre-coated with transferrin also demonstrate

cellular uptake with HeLa, SNB19 and STO cells, albeit at different concentrations but with

similar trends and further suggest that uptake is likely due to a clathrin mediated process.

Authors put forward the importance of competition between thermodynamic driving

force for wrapping (how the membrane encloses a particle which involve factors such as ratio

of adhesion and membrane stretching, the membrane’s bending energy) and the receptor

diffusion kinetics (kinetic of recruitment of receptors to the binding site), for nanoparticle cell

uptake. Consistent with previous results, they found that spherical gold nanoparticles with

size of 50 nm have the greatest degree in cellular uptake; smaller nanoparticles needed to

cluster together to go in and larger nanoparticles having a longer wrapping time due to the

slower receptor diffusion kinetic. However, they proposed, as the 50 nm nanoparticle can

enter cells as single nanoparticle, that vesicles must fuse later on in the cell after entrance.

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Further, detailed TEM studies have shown that nanoparticles are trapped in endosomes before

being fused with lysosomes for processing.

Specific mechanisms for nanoparticles cell clearance were conducted with gold

nanoparticles precoated with transferrin. Studies were performed with STO, HeLa and SNB19

cell types. Removal of transferrin-coated gold nanoparticles was linearly related to size,

which was different than the uptake process. Nanoparticles about to be removed from cells

appeared to be localized in late endosomes or lysosomes. Smaller nanoparticles were found to

exocytose faster in contrast to larger nanoparticles. Smaller nanoparticles will have less-

receptor ligand interactions and the authors speculated that fewer receptor-ligand interactions

led to a lower overall binding constant and hence, the transferrin coated nanoparticles could

be released more easily.

Despite a huge amount of work performed by Chithrani et al., to deeply understand the

effects of gold nanoparticles size, shape and surface coating on intracellular uptake, transport

and subcellular nanoparticles localization, no studies have been published, to our knowledge,

to evaluate their efficacy when activated by ionizing radiations.

Gold nanoparticles toxicity

It is generally admitted that gold nanoparticles are biocompatible and nontoxic.

However, in vitro and in vivo studies have recently raised the potential toxicity of gold

nanoparticles. In the following, a literature survey on spherical gold nanoparticles is

presented, excluding gold nanoparticles with size of 1.4 nm which have shown to enter into

the nucleus of cells and to interact with DNA inducing significant toxicity even at very low

level. (Pan et al., 2007; Tsoli et al., 2005)

In vitro studies

Two papers (Yan et al., 2009 and Connor et al., 2005) present in vitro cytotoxicity

studies with gold nanoparticles with size of 3.7 nm (HeLa cell line) and ∼ 4 nm, 12 nm and 18

nm (K562 leukemia cell line). Nanoparticles were functionalized with different surface

treating agents. As a brief summary, the data report that gold nanoparticles are taken up by

cells but do not cause acute cytotoxicity. Gold nanoparticles with size of 3.7 nm modified

with PEG were found into the nucleus of HeLa cells upon exposure for 24 hours. Following

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those results, Pernodet et al., 2006, have focused in greater details on citrate / gold

nanoparticles with size of 14 nm and investigated further the effects of gold nanoparticles on

cell proliferation, morphological structure, spreading, migration and protein synthesis. In the

long run, these factors may be more dangerous as they trigger the growth of defective living

tissues. Authors studied these effects on human dermal fibroblast cells and focused on the

effects of nanoparticles on individual living cells. The major conclusions of their study is that

(1) 14 nm citrate / gold nanoparticles can easily cross cell membranes and accumulate into

vacuoles (nanoparticles cells clearance was not discussed). The entry of the nanoparticles

appears to be not immediately detrimental to cell function, but rather the formation of an

unusually large number of vacuoles probably triggers a serie of secondary events, which

eventually hinders other processes; (2) the presence of gold nanoparticles, in a concentration

dependant manner, is responsible for abnormal actin filaments (either a depolymerization

process or a lack of actin-fiber formation) and ECM constructs in dermal fibroblast. These, in

turn, are shown to decrease cell proliferation, adhesion and motility. Further studies are in

progress to identify the specific genes expressed and the protein produced. Still, on similar

gold nanoparticles, Khan et al., 2007 have performed cytotoxicity assays, uptake studies and

gene expression profiling on HeLa cells. Gross changes in gene-expression patterns were not

seen after uptake of 18 nm gold nanoparticles into the human cell line HeLa. In spite of their

ability to bind to serum proteins non specifically, gold nanoparticles did not induce the

splicing of xbp1 mRNA, which is a marker of unfolded protein response.

Patra et al., 2007 investigated cells specific response to citrate / gold nanoparticles

with size of 33 nm. They report that gold nanoparticles-induced death response, in a

concentration dependant manner, in human carcinoma lung cell line A549 but not on the 2

others cell lines tested, BHK21 (baby hamster kidney) and HepG2 (human hepatocellular

liver carcinoma) which remain unaffected by the presence of gold nanoparticles. Authors

confirmed that A549 cytototoxicity is induced by the gold nanoparticles. Gradual increase in

gold nanoparticles concentration induces a proportional cleavage of poly(ADP-ribose)

polymerase. The programmed nature of the death response is implied, because such cleavage

follows activation of caspases.

In vivo studies

Chen et al., 2009 studied in vivo toxicity of naked gold nanoparticles with size of 3, 5,

8, 12, 17, 37, 50 and 100 nm. Naked nanoparticles were prepared by seed process in presence

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of citrate and NaBH4. To our understanding, citrate is believed to act as complexing agent.

Naked gold nanoparticles were injected intraperitoneally into BALB/C mice at dose of 8

mg/kg/week. Gold nanoparticles of 3, 5, 50 and 100 nm did not show harmful effects;

whereas gold nanoparticles ranging from 8 to 37 nm induced severe sickness in mice.

Pathological examination of the major organs of the mice in the diseased groups indicated an

increase of Kupfer cells in the liver, loss of structural integrity in the lungs and diffusion of

white pulp in the spleen. The pathological abnormality was associated with the presence of

gold nanoparticles at the diseased sites. Modifying the surface of gold nanoparticles by

incorporating immunogenic peptides ameliorated their toxicity. This reduction in the toxicity

was associated with an increase in the ability to induce antibody response. Gold nanoparticles

larger than 50 nm were non toxic to mice, which could be interpreted as a diffusion-restricted

region. The non toxic effect of gold nanoparticles smaller than 5 nm could be explained by the

increase in antibody response that enhanced the scavenging effect. Further, authors stipulated

that urinary excretion may play an important role to remove gold nanoparticles under 5 nm in

their model.

Interestingly, Hainfeld et al., 2006 have performed studies with gold nanoparticles of

1.9 nm (gold nanoparticles from Nanoprobes), with the aim to develop the use of such

nanoparticles as new X-ray contrast agents. With 10 mg Au/ml initially in the blood, mouse

(BALB/C mice bearing EMT-6 subcutaneous mammary tumours) behaviour was

unremarkable and neither blood plasma analytes nor organ histology revealed any evidence of

toxicity 11 days and 30 days after i.v injection. The highest tissue gold concentration 15 min

after injection was in the kidney (10.6 ± 0.2 % of the injected dose per gram of measured

tissue – % id/g), followed by tumour (4.2 ± 0.4 % id/g), liver (3.6 ± 0.3 % id/g) and muscle

(1.2 ± 0.1 % id/g). However, whole body gold clearance was 77.5 ± 0.4 % of the total injected

dose after 5 hours. Muscles and blood were almost gold free 24 hours after injection, while

kidney, liver and tumour kept constant gold concentration. Here, the rapid and high level of

gold nanoparticle clearance could explain the absence of gold nanoparticle toxicity.

Cho et al., 2009 carried out in vivo toxicity study using 13 nm-sized gold nanoparticles

coated with PEG, using thiol-terminated PEG (MW 5000). It is worth mentioning that the

thiol (SH) function is known to graft strongly to the surface of gold nanoparticles and it is not

expected that such surface coating will be displaced by serum proteins. However, serum

proteins may interact with the coating. Quite surprisingly, the gold nanoparticles were seen to

induce acute inflammation and apoptosis in the liver. The nanoparticles were found to

accumulate in the liver and spleen for up to 7 days after i.v injection. In addition, TEM

- 120 -

showed that numerous cytoplasmic vesicles and lysosomes of liver Kupfer cells and spleen

macrophages contained PEG-coated gold nanoparticles. Although the transient inflammatory

responses were negligible for the toxicity of 13 nm PEG-coated gold nanoparticles, apoptosis

was important effects induced by treatment of 13 nm PEG-coated gold nanoparticles.

Despite the establishment of in vitro and in vivo efficacy using gold nanoparticles in

combination with ionizing radiations gold nanoparticles toxicity studies strongly suggest that

careful scrutiny of the in vivo and in vitro toxicities of gold nanoparticles is required even if

gold nanoparticles have previously shown to have limited or no toxicity at the cellular level.

3. Enhancement of radiation activity of nanoXrayTM

3.1. “Endosomes Bioavailability as Key Parameter for Optimal Efficacy of Radiation Enhancer Nanoparticles on Human Colon Cancer Cells”

Virginie Simon, Ping Zhang, Laurence Maggiorella, Agnès Pottier, Elsa Borghi, Laurent Levy, Julie Marill ; In Preparation

Medicine is now using physics every day to treat cancer patients. Nanomedicine can

help clinicians in delivering safer and more efficacious treatments by shifting the intended

effect from the macroscopic to the subcellular level.

NBTXR3 is designed to resolve cancer therapy’s biggest drawback: destruction of

healthy tissue and its subsequent deleterious side effects when a high dose of X-ray is

necessary. Indeed, high dose of X-ray cause burns and necrosis in tissue around reconstructive

wires in mandibular cancer patients after radiation treatments (Castillo et al., 1988). The

concept of using high Z materials for dose enhancement in cancer radiotherapy was advanced

over 20 years ago by Matsudaira et al., 1980, who measured a radioenhancing effect of iodine

on cultured cells. Nath et al., 1990 incorporated iodine into cellular DNA with

iododeoxyuridine in vitro, and found a radiation enhancement of around 3. Regulla et al.,

1998 showed a physical dose enhancement factor (DEF) around 100 within a range of 10 µm

and a biological enhancement factor of up to 50 for fibroblast monolayers irradiated on a gold

foil. Herold et al., 2000 injected 1.5-3 µm gold particles directly into a tumour followed by

irradiation and found that excised cells had reduced plating efficiency.

- 121 -

NanoXrayTM plateform concept is the design and development of therapeutic metal

oxide nanoparticles which core is hard and highly inert and constitutes the matter for

nanoparticles – ionizing radiation interactions triggering emission of electrons losing energy

and the subsequent creation of free radicals. NBTXR3 nanoparticles have an on-off mode of

action. When the beam of ionizing radiation is stopped the nanoparticles return to the basal

inert crystalline status.

NBTXR3 nanoparticles are homogenous in size with a mean hydrodynamic diameter

of approximately 70 nm. Such range seems relevant for a superior cellular penetration as

shown by Chithrani et al., 2006 with gold nanoparticles. Stability in physiological media is

achieved by coating the nanoparticles with a surface treating agent, which ensures the stability

of the nanoparticles in physiological fluids.

This paper describes for the first time, in vitro studies with NBTXR3 oxide metallic

nanoparticles. This article shows NBTXR3 nanoparticles trafficking mechanism at cellular

level, using complementary and comparative technical methods.

- 122 -

ARTICLE -I-

Endosomes Bioavailability as Key Parameter for Optimal Efficacy of

Radiation Enhancer Nanoparticles on Human Colon Cancer Cells

Virginie Simon, Ping Zhang, Laurence Maggiorella, Agnès Pottier, Elsa Borghi, Laurent Levy, Julie Marill,

(In Preparation)

- 123 -

3.2. Discussion

The goal of this work was to study in vitro enhancement of radiation cytotoxicity in

HCT 116 colon cancer cells by local activity of NBTXR3 nanoparticles.

Viability assays (4-[3-(4-Iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene

disulfonate for WST-1) and clonogenic assays were performed to evaluate NBTXR3

nanoparticles effect on cell survival.

Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) was used for NBTXR3

nanoparticles quantification at the cellular level (nanoparticles bound to the cells membrane

and/or internalized). Indeed, ICP-MS presents the advantage to quantify NBTXR3

nanoparticles at cellular level, but has the disadvantage to not differentiate between the total

amount of nanoparticles internalized or bound to cell membranes.

TEM semi quantitative analysis was investigated for the first time in order to establish

nanoparticles trafficking at cellular level, but also to define optimum schedule to achieve

significant tumour cells destruction and to visualize the local effect of activated nanoparticles

on cells. The aim of visualizing and analyzing ultrathin sections as a prelude to TEM is to

reveal the internal structure of HCT 116 cells at a sufficiently high level of resolution. It is

also important to remember that the number of images observed and counted on section

planes is crucial for the robustness of semi quantitative analysis.

Through all those assays, the ratio between the number of NBTXR3 nanoparticles and

cells was kept constant. Hence, we are strongly confident in our results comparison and

conclusions between all those assays.

Taking together those complementary approaches, NBTXR3 nanoparticles mechanism

of action on cell is proposed. Most appropriate schedules could be defined for nanoparticles

use.

We have demonstrated NBTXR3 nanoparticles dose effect on cell viability. Statistical

difference on cell viability between radiotherapy control alone and activated NBTXR3

nanoparticles was established for cells incubated with nanoparticles at a concentration of 100

µM and beyond. DEF estimates the increase in radiation cells response triggered by NBTXR3

nanoparticles, in comparison to the irradiation control alone. DEF of 3.45 and 4.88, for

NBTXR3 concentration of 400 µM and 4 Gy irradiation, were obtained for colon HT-29 and

HCT 116 cells respectively. Local intracellular activity of NBXT3 nanoparticles was proved.

Correlation was observed between NBTXR3 nanoparticles cell uptake, cytoplasmic

localization and efficacy. Study of interaction between NBTXR3 nanoparticles and HCT 116

- 124 -

cell in term of cell trafficking was performed for the first time. Based on the analysis of TEM

and SEM images, we have shown that NBTXR3 nanoparticles penetrated the cells by

endocytosis according to physical interaction of NBTXR3 with cell membrane. Nanoparticles

assemble as clusters within endosomes in cytoplasmic compartment. Chithrani et al., 2007

compared the exocytosis of spherical gold nanoparticles, with 3 differents sizes, on HeLa

cells. The authors showed that after 8 hours of incubation, the fraction of gold nanoparticles

with size of 14, 50 and 74 nm exocytosed was equal to 40%, 20% and 10% respectively. In

contrast, NBTXR3 nanoparticles were demonstrated to have long residence within the cell

cytoplasm.

Although most studies suggest that DNA is damaged indirectly by hydroxyl radicals,

electrons can also cause damage to DNA directly, as illustrated in a recent study in which

low-energy electrons emitted from metal films were found to cause DNA strand breaks

directly (Carter et al., 2007). In our study, local effect of activated NBTXR3 nanoparticles on

cells is observed on both cell nucleus and cell organelles. Increase of the number of

micronuclei formation was observed for irradiated cells incubated with NBTXR3

nanoparticles when compared to irradiation control alone. Also, significant cells

disorganization was observed for cells incubated 24 hours with NBTXR3 nanoparticles prior

irradiation.

In vivo efficacy published results, with gold nanoparticles (Hainfeld et al., 2004;

Chang et al., 2008), have only been performed with one single and high irradiation dose (25-

30 Gy delivered). Interestingly, our in vitro results showed that a dose delivered of 2 Gy

(using a kilovoltage X-ray beam) was sufficient to induce a significant enhancement of

radiation cytotoxicity, for HCT 116 cells incubated with NBTXR3 nanoparticles.

Furthermore, preliminary in vivo efficacy on mice bearing HCT 116 tumour xenografted was

performed with fractionated irradiation and demonstrated very promising tumours eradication.

This is an important consideration, because sparing dose to healthy tissue is of primary

concern in all radiation therapy procedures.

Next experimental studies should focus on a deeper cell organelles characterization:

structures like lysosomes and autolysosomes should be confirmed using specific fluorescence

markers. For instance, colocalization of NBTXR3 nanoparticles and lysosomes within live

HCT 116 cells should be evaluated using specific alexa-647 lysosomes marker

(excitation/emission at 550/568 nm).

Furthermore, HCT 116 cells TEM imaging could be investigated at 4°C to precise the

endocytosis pathway.

- 125 -

Finally, studies should be investigated in others tumour cell lines with biologic

specificities: their origin (epithelium and mesenchymal origin, different compartmental

structure), human healthy versus malignant tumour cells, radiosensitivity characteristics

(radiosensitive or radioresistant cancer types).

4. Conclusion

Gold nanoparticles are the single reference in the literature of metallic nanoparticles,

activated by ionizing radiations, to treat cancers. Published studies present efficacy and

toxicity results, both in vitro and in vivo, which support foundation for a rational of

development of gold nanoparticles in the clinic.

Concerning the in vivo toxicity, Chen et al., 2009 showed that naked gold

nanoparticles with size ranging from 8 to 37 nm induced severe sickness in mice. Further,

Cho et al., 2009 observed that 13 nm-sized gold nanoparticles coated with PEG, using thiol-

terminated polyethylene glycol, a strong anchoring surface treating agent, induced acute

inflammation and apoptosis in the liver.

Concerning efficacy, only two preclinical studies are reported (Hainfeld et al., 2004;

Chang et al., 2008). Of note, very small size of gold nanoparticles was used for those studies

(1.9 nm and 13 nm respectively). In Hainfeld study report, nanoparticles are irradiated 2 min

following gold nanoparticles i.v. injection. Because of the gold nanoparticles size (1.9 nm),

gold nanoparticles are cleared from tumour very rapidly. Gold nanoparticles with increased

nanoparticle size around 50-100 nm are believed to be more attractive for clinical applications

when considering the nanoparticles cell uptake (Chithrani et al., 2006) and the nanoparticles

retention within tumour cells - the smaller nanoparticles are found to exocytose faster and in

higher proportion (Chithrani et al. 2007). Furthermore, in these two in vivo studies, a very

high dose of irradiation was delivered (30 and 25 Gy respectively) in a single fraction, which

seems quite unrealistic for clinical application.

Fractionated radiation therapy constitutes the standard of care for cancer treatment.

Recent randomized trials have confirmed that hypofractioned whole-breast irradiation (as for

other cancer diseases) is equivalent to more conventional whole breast irradiation with respect

to local recurrence and cosmetic outcome. For instance, for whole breast irradiation after

breast conserving surgery a 1.8 to 2 Gy daily fractions is given 5 times a week to a total dose

of 45 to 50 Gy over 5 weeks with the optional addition of a boost to the primary site of 10 to

16 Gy in 5 to 8 daily fractions over 1 to 1.5 weeks. Through empiric observation, it has

- 126 -

become clear that the therapeutic ratio, the balance between tumour cell kill and normal tissue

damage, is affected not only by fraction size but also the total dose of radiation and in some

instances overall treatment time and the volume of tissue irradiated (Whelan et al., 2008).

NBTXR3 nanoparticles development has included X-ray standard protocols, as

routinely used in the clinic for radiotherapy.

Despite of the establishment of in vitro and in vivo efficacy gold nanoparticles use in

the clinic should present some limitations – such as the use of high energy dose delivered in

one fraction – which suggest that gold metallic nanoparticles activated by ionizing radiations

are delaying for their application to treat cancer. Furthermore, toxicity studies strongly

suggest that careful scrutiny of the in vivo and in vitro toxicities of gold nanoparticles is

required even if gold nanoparticles have previously shown to have limited or no toxicity at the

cellular level.

In this work, we have demonstrated the NBTXR3 efficacy in clonogenic tests both in

HCT 116 and HT-29. No significant clonogenic toxicity of NBTXR3 was observed in these

cell lines. Cell viability assays were also performed on HCT 116 following 2 Gy irradiation

with significant difference of efficacy when compared to radiotherapy alone.

NBTXR3 are internalized by endocytosis. TEM analysis studies suggest that NBTXR3

nanoparticles stay within the cells cytoplasm. Furthermore, NBTXR3 nanoparticles

trafficking at cellular level, evaluated through viability assays and NBTXR3 nanoparticles

quantification, support the hypothesis that nanoparticles have long residence within the cells

cytoplasm, in late endosomes.

Furthermore, NBTXR3 nanoparticles have demonstrated to bring survival benefit in

mice bearing HCT 116 tumour xenografted (data not shown). The time of NBTXR3

nanoparticles residence within tumour was demonstrated to be longer than 15 days.

As opposed to gold nanoparticles trafficking at cellular level reports and currently

published in vivo efficacy studies, NBTXR3 results demonstrate a real advantage for use with

standard radiotherapy protocols.

- 127 -

Pp IX silica nanoparticles create differential

biodistribution between tumour and healthy tissues

in terms of tumour uptake and subcellular

localization: photodynamic therapy therapeutic

window enlargement

- 128 -

1. Photodynamic therapy mechanism of action

Three components are essential in the PDT reaction: the photosensitizer, the light with

appropriate wavelength and the presence of oxygen. The interaction among these three

constituents is the main part of the treatment effect.

A photosensitizer is a chemical compound that can be excited by light of a specific

wavelength. This excitation uses visible or near-infrared light. In photodynamic therapy,

either a photosensitizer or the metabolic precursor of one is administered to the patient. The

tissue to be treated is exposed to light suitable for exciting the photosensitizer. Usually, the

photosensitizer is excited from a ground singlet state to an excited singlet state. It then

undergoes intersystem crossing to a longer-lived excited triplet state. One of the few chemical

species present in tissue with a ground triplet state is molecular oxygen. When the

photosensitizer and an oxygen molecule are in proximity, an energy transfer can take place

that allows the photosensitizer to relax to its ground singlet state, and to create an excited

singlet state oxygen molecule (Josephsen et al., 2008; Figure 37). Singlet oxygen is a very

aggressive chemical species and will very rapidly react with any nearby biomolecules. It is

important to note that although PDT results in singlet oxygen species, these reactive radicals

are short-lived (µs), with a radius of action of only 0.01 µm, and therefore have very low

mutagenic potential for DNA damage. The specific targets depend heavily on the

photosensitizer chosen, a point detailed in a later paragraph. Ultimately, these destructive

reactions will kill cells through apoptosis or necrosis.

Figure 37: Modified Jablonski energy diagram (Josefsen et al., 2008)

- 129 -

2. Photosensitizer: light excitation

2.1. Optimum for superficial tumours: light penetration in tissues

To carry out PDT successfully in vivo, it is necessary to ensure that sufficient light

reaches all the diseased tissues. This involves understanding how light travels through various

tissues and the relative effects of light absorption and scattering. In PDT, it is important to be

able to predict the spatial distribution of light within th e target tissue. Light is either

scattered or absorbed when it enters tissues and the extent of both processes depends on the

tissue type and light wavelength utilized (Robertson et al., 2009). The light transmission has

been experimentally measured for various tissues as a function of the sample thickness and of

the wavelength of the incident light. Interestingly, the maximum fluence rates occur below the

surface. The position of this peak shifts more and more into the tissue with increasing

wavelength. Although large variations in the absolute values arise, owing to differences in the

biological material, the relative trend of the spectral dependence is quite similar. Most tissues

show an increased transparency towards higher wavelengths with a maximum penetration

depth between 700 and 800 nm (Ochsner et al., 1996). This is also involved because the

biological tissue is inhomogeneous and the presence of microscopic inhomogeneities

(macromolecules, cell organelles, organized cell structure, interstitial layers) makes biological

tissues turbid. Multiple scattering within a turbid medium leads to spreading of a light beam

and loss of directionality. Absorption is largely due to endogenous tissue chromophores such

as hemoglobin, myoglobin and cytochromes. The light source for PDT must exhibit suitable

spectral characteristics which coincide with the maximum absorption wavelength range of the

photosensitizer applied in order to generate enough ROS to produce a cytotoxic effect. Thus,

for efficient photodynamic laser mediated therapy, light excitation between 700 and 800 nm is

preferred to allow an increased penetration depth with minimal light scattering while

expecting maximum photosensitizer activation, with the most tissue destruction occurring

within the targeted tissue (Robertson et al., 2009).

Photosensitizer from porphyrin family, and their lead product Photofrin�, are the first

generation of photosensitizer developed for PDT. On account of their highly conjugated

skeleton, porphyrins have a characteristic ultra-violet visible spectrum which consists of an

intense Soret band at approximately 400 nm, followed by four longer weak absorptions (� >

20 000 lmol−1cm−1) Q bands (500, 540, 570 and 630 nm respectively) (Figure 38).

- 130 -

Figure 38: Typical porphyrin absorption spectrum and modification of Jablonski energy diagram (Josefsen et al., 2008)

Despite the weak absorption properties of Photofrin� in the near-infrared region, the

630 nm absorption peak was selected for light excitation in order to penetrate tissues.

However, for light in the near infrared region (630 nm), a “rule of thumb” is that about 2-5

mm of tissue can be treated, depending on the optical properties of the tissue, and the

dosimetry used (Wang et al., 1999; Figure 39). That is why the most common application of

PDT was intended so far, for superficial tumours treatment.

Figure 39 : Light penetration deep in tissues function wavelength: limitation of PDT (Croisy et al., 2005)

VeinArtery VeinArtery

- 131 -

2.2. Chemistry improvement: second, third generation of photosensitizer

While the major disadvantages associated with the first generation photosensitizer

hematoporphyrin (HpD) and Photofrin® have not prevented the treatment of some cancers

and other diseases, they have markedly reduced the successful application of these

photosensitizers to a wider field of disease.

It is today commonly admitted that a well-designed photosensitizer should exhibit the

following properties: long-wave absorption, good singlet oxygen quantum yield, quite simple

structure and easy synthesis process route, good in vivo stability and reduce adverse effects.

This area of research led to the development of a second generation of photosensitizers,

designed to minimise the drawbacks of the first generation photosensitizer. A number of new

sensitizers were therefore developed to overcome these short comings such as 5-

Aminolaevulinic acid (5-ALA), Verteprofin, Purlytin, Foscan®, Lutex, ATMPn, Zinc

phthalocyanine CGP55847 and Naphthalocyanines (Figure 40).

Figure 40: Summary of a collection of different photosensitiser types and their absorption data (Josefsen et al., 2008)

- 132 -

A number of the second generation photosensitizer described earlier contains a

chelated central metal ion. Expanded porphyrins have a larger central binding cavity,

increasing the number of potential metals it can accommodate. The metallation of a number of

these chromophores has generated a variety of photosensitizers with improved photophysical

properties. The effectiveness of these metallo-photosensitizers depends largely (but not

definitively) on the nature of the coordinated central metal ion. Chromophores chelated to

diamagnetic transition metals and lanthanide ions have shown the greatest potential as

photodynamic agents, a consequence of the heavy metal effect enhancing the rate of

intersystem crossing. As a result, a number of these metallated tetrapyrrole-based macrocycles

are currently photosensitizers of choice, particularly the zinc (II), aluminium (III), and tin (IV)

complexes. Ochsner et al (1996) showed that the Zinc(II)-phthalocyanine absorption

maximum lies at about 670 nm and its associated extinction coefficient is at least 60 times

larger than that of Photofrin® at its treatment wavelength (630 nm). The greater the

extinction coefficient value, the smaller is the drug dosage required to induce a cytotoxic

response and the risk of provoking systemic toxic reactions is clearly diminished. The longer

absorption wavelength correlates with a higher penetration depth. Since the skin

phototoxicity is dominated by the absorption of sunlight, a shift of the main absorption peak

to higher wavelengths and a smaller overlap of the absorption spectrum of the sensitizer with

the sunlight emission spectrum are expected to reduce the risk of inducing phototoxic

reactions.

Despite significant improvement in the design of photosensitizer for a more efficient

interaction with laser light and a subsequent enhancement of ROS generation, many

drawbacks are pendant which had reduced their development in the clinic. Indeed, most of the

developed photosensitizers are hydrophobic and aggregate easily under physiological

condition which results in the difficulty in preparing pharmaceutical formulations that enable

parenteral administration. Moreover, even with hydrophilic ones, the accumulation selectivity

to specific cells or tissues is still usually too low for clinical use.

Works have recently focused on designing a third generation of photosensitizer, with

specific designed to effect greater selectivity and specificity toward targeted tissues.

Maillard and his colleagues (2007) have screened 17 new potential products for PDT

application. In vitro photocytotoxicity of hydrophenylporphyrins and chlorines and their

glycoconjugated derivated (glucose/galactose/mannose) were tested both in retinoblastoma

cell line (Y79) and HT-29 cell line. The goal was to determine whether the covalent coupling

- 133 -

of glycoside residues to the macrocycle would lead to a significant enhancement in PDT

retinoblastoma treatment for the derived conjugates relative to the parent free OH compounds.

These results showed that the nature and anomeric configuration of the sugar component are

important for both in vitro cytotoxicity and efficacy. For instance, glycosylated porphyrins in

which the galactose component is separated from the p-hydroxyphenyl motif by a

diethyleneglycol linker display a 10 times greater photocytoxicity for � configuration

compared by � one.

At present, the next generation of photosensitizer is the development of nanocarriers.

Nanocarrier is intended to: (1) protect a drug from degradation, (2) enhance drug absorption

by facilitating diffusion through epithelium, (3) modify pK and drug tissue distribution

profile, and (4) improve intracellular penetration and distribution (Couvreur et al., 2006).

Those nanocarrier systems are developed to induce greater selectivity and specificity when

compared with the “free” photosensitizer molecule in order to increase the therapeutic effect.

2.3. Presentation of approved photosensitizers and current research

2.3.1. Porphyrin, texaphyrin and chlorin photosensitizers in clinics

Porphyrins are a heterocyclic aromatic ring made from four pyrrole subunits joined

on opposite sides through four methine links. Most current clinical porphyrin photosensitizers

derivate are Photofrin®, Levulan®, Metvix® and Visudyne®.

Photofrin® was the first drug for PDT to receive regulatory approval for the treatment

of obstructing oesophageal cancer in 1993 in Canada and in 1995 in US. Photofrin® is

commercially available from Axcan Pharma, Inc. and has the longest clinical history and

patient track record (Figure 41). The photosensitizer consists of about 60 compounds and

therefore it is difficult to reproduce its composition. It is actually a proprietary combination of

monomers, dimers, and oligomers. It should be noted that the first reports of PDT success in

patients using Photofrin® were on bladder cancer treatment (Allison et al., 2004).

- 134 -

Figure 41: Type of cancer and approved PDT drugs

Texaphyrins are expanded, porphyrin-like macrocycles which complex large metal

cations. The dyes have a high absorbance peak in the near infrared, 730-770 nm. Most current

clinical texaphyrin photosensitizer derivate is Antrin®.

Chlorins constitute a group of molecules very similar to porphyrins. Chlorins can also

be synthesized from porphyrins. In contrast to porphyrins, chlorins have the strongest

absorption peaks in the red part of the spectrum, which give the compounds a green colour. In

comparison to the porphyrin structure, the chlorin has at least one double bond missing in the

pyrrole rings. Most current clinical chlorin photosensitizers derivate are Foscan®, LS11 and

Photochlor (Figure 42).

Disease Drug Country

Pre

-can

cer

Can

cer

Actinic keratosis

Barett’s oesophagus

Cervical dysplasia

Basal-cell carcinoma

Cervical cancer

Endobroncheal cancer

Oesophageal cancer

Gastric cancer

Head and neck cancer

Papillary bladder cancer

Metvix ®

Photofrin®

Photofrin®

Photofrin®

Photofrin®

Foscan®

Photofrin®

Levulan, Metvix®

Photofrin®

Photofrin®

EU

Japan

Canada, Denmark, Finland, France, Germany, Ireland, Japan, The Netherlands, UK, USA

Canada, Denmark, Finland, France, Ireland, Japan, TheNetherlands, UK, USA

Japan

EU

Canada

EUEU, USAJapan

Disease Drug CountryP

re-c

ance

rC

ance

r

Actinic keratosis


Cervical dysplasia


Cervical cancer


Oesophageal cancer

Gastric cancer



Metvix ®

Photofrin®

Photofrin®

Photofrin®

Photofrin®

Foscan®

Photofrin®

Levulan, Metvix®

Photofrin®

Photofrin®

EU

Japan



Japan

EU

Canada

EUEU, USAJapan

v

v

v

Disease Drug Country

Pre

-can

cer

Can

cer

Actinic keratosis


Cervical dysplasia


Cervical cancer


Oesophageal cancer

Gastric cancer



Metvix ®

Photofrin®

Photofrin®

Photofrin®

Photofrin®

Foscan®

Photofrin®

Levulan, Metvix®

Photofrin®

Photofrin®

EU

Japan



Japan

EU

Canada

EUEU, USAJapan

Disease Drug CountryP

re-c

ance

rC

ance

r

Actinic keratosis


Cervical dysplasia


Cervical cancer


Oesophageal cancer

Gastric cancer



Metvix ®

Photofrin®

Photofrin®

Photofrin®

Photofrin®

Foscan®

Photofrin®

Levulan, Metvix®

Photofrin®

Photofrin®

EU

Japan



Japan

EU

Canada

EUEU, USAJapan

v

v

v

- 135 -

Figure 42: List of the current clinical photosensitizers and their manufacturers

2.3.2. Others trends

Development of new photosensitizers as well as the optimization of PDT protocols,

such as fractionation of light or drugs, well-designed clinical trials which involve selectively

localized photosensitizers and convenient light sources will improve the prospects for the use

of PDT in cancer.

In addition, current research topics are emerging with the ambition to enlarge the

possibility for PDT application, by either increasing the photosensitizer light interaction using

already approved photosensitizers (two photon absorption concept), or by bringing additional

modalities to current treatments such as combined diagnostic and PDT (theranostic

application) or even combined therapy.

� Two photon absorption (TPA): One of the most relevant remaining limitation of

PDT is the limited light penetration within tissues. TPA-induced excitation of photosensitizer

is a promising approach for increasing light penetration because it makes it possible to use

two photons of lesser energy (higher wavelength) to produce an excitation that would

“normally” be produced by the absorption of a single photon of higher energy (lower

wavelength). Indeed, appropriate photosensitizers can simultaneously absorb two photons of

lower energy, which makes excitation possible in the near IR region, thereby avoiding

Platform Drug Substance Manufacturer

Porphyrin

Texaphyrin

Chlorin

Photofrin®

Levulan®

Metvix®

Visudyne®

Antrin®

Foscan®

LS11

Photochlor

HpD

ALA

M-ALA

Vertiporfin

Lutexaphyrin

Temoporphin

Talaporfin

HPPH

Axcan Pharma, Inc.

DUSA Pharmaceuticals, Inc.

PhotoCure ASA

Novartis Pharmaceuticals

Pharmacylics

Biolitec Pharma Ltd.

Light Science

RPCI

Platform Drug Substance Manufacturer

Porphyrin

Texaphyrin

Chlorin

Photofrin®

Levulan®

Metvix®

Visudyne®

Antrin®

Foscan®

LS11

Photochlor

HpD

ALA

M-ALA

Vertiporfin

Lutexaphyrin

Temoporphin

Talaporfin

HPPH

Axcan Pharma, Inc.

DUSA Pharmaceuticals, Inc.

PhotoCure ASA

Novartis Pharmaceuticals

Pharmacylics

Biolitec Pharma Ltd.

Light Science

RPCI

- 136 -

wasteful tissue absorption or scattering and allowing a deeper penetration of light into the

tissue. Kim et al., 2007 describ the synthesis of organically modified silica nanoparticles in

which 2-devinyl-2-(1-hexyloxyethyl) pyropheophorbide (HPPH) and an excess of 9,10-bis[4’-

(4’’-aminostyryl)styryl]anthracene (BDSA), a highly two-photon-active molecule acting as an

energy donor, were co-encapsulated. HPPH absorption in nanoparticles had significant

overlap with the fluorescence of BDSA aggregates, which enabled an efficient energy transfer

through fluorescence resonance energy transfer (FRET) mechanisms. After indirect two-

photon excitation (850 nm), the authors provided evidence that the energy of the near-IR light

was sufficiently upconverted by BDSA aggregates to be able to excite HPPH, leading to

formation of 1O2. The intracellular FRET efficiency was estimated as being ~36%. Drastic

changes in the morphology of the cells were observed, which were indicative of impending

death as a result of photocytotoxicity of HPPH.

� Diagnostic and therapy: Lai et al., 2008 present a highly uniform Fe3O4/SiO2

core/shell nanoparticles functionalized by phosphorescent iridium (Ir) complexes to form

Fe3O4/SiO2 Ir. Authors have strategically designed a hydrophilic Ir complex suited for

simultaneous phosphorescence imaging and 1O2 production. MTT assays on HeLa cells

showed that almost 100% of the cells were viable without activation, even after 24 hours of

incubation with 100 mg/ml of Fe3O4/SiO2 Ir. Cellular uptake was dose responsive and the

uptake could be imaged by MRI even at the lowest concentration (detection maximal of 104

cells). In this study, Fe3O4/SiO2 Ir composite demonstrated its potential in multiple

applications, the magnetic core providing the capability for MRI and the Ir complex greatly

enhancing the spin-orbit coupling for phosphorescent labeling, and simultaneous 1O2

generation inducing apoptosis.

� Combined therapy: Gu et al., 2005 report the synthesis, characterization, and

cellular uptake of the conjugate of porphyrin and iron oxide nanoparticles, which may lead to

a bimodal anticancer agent which could be used in the combinational treatment of PDT and

HT. The HeLa cells were incubated with the Fe3O4-porphyrin nanoparticles at 37 °C for 5

hours without showing observable dark toxicity. When cells were exposed to irradiation

during 10 min the cells changed their conformation and went in apoptosis cell death. Cells

were then observed under a fluorescence microscope. Fe3O4-porphyrin nanoparticles are

uptaken by the HeLa cells, likely as the result of endocytosis into the cytoplasm. Also HT

therapy was not studied, the thermal stability of the nanoparticle was performed. Following

- 137 -

nanoparticles boiling in a water-methanol solution for 30 min, no loss of porphyrin

fluorescence spectrum was detected, suggesting a good tolerance of the nanoparticles and a

subsequent eligible candidate for HT.

3. Systemic administration of photosensitizer for very localized treatment

3.1. Half life of generated ROS and distance for ROS effect: subcellular localization of the photosensitizer is required

PDT is an anticancer approach, which achieves tumour cell destruction after light

excitation of the photosensitizer within a well restricted area. The high selectivity of tumour

site cytoxicity is based on the application of the external energy on the cancer anatomical

localisation for agents usually administered by systemic route and with broad body

distribution.

Also, these sites of photodamage may reflect the localisation of the photosensitiser in

the cell, in subcellular organelles and close to biomolecules. A variety of cellular components

such as amino acids (particularly cysteine, histidine, tryptophan, tyrosine and methionine),

nucleosides (mainly guanine) and unsaturated lipids can react with singlet oxygen. The

diffusion distance of singlet oxygen is relatively short (about 0.01 µm), therefore the

photosensitiser must associate intimately with the substrate for efficient photosensitisation to

occur.

Many factors determine the cellular targets of photosensitizer. The incubation

parameters and mode of delivery as well as the chemical nature of the photosensitiser can all

influence subcellular localisation, creating a number of potential targets for photodamage. In

cell culture studies with porphyrin based photosensitizer, short incubation times (up to 1 hour)

prior to illumination leads primarily to membrane damage whereas extended incubation

periods followed by light exposure results in damage to cellular organelles and

macromolecules.

Hydrophobic (lipophilic) compounds preferentially bind membranes and will target

structures such as the plasma membrane, mitochondria, lysosomes, ER and the nucleus.

Oxidative degradation of membrane lipids can cause the loss of membrane integrity, resulting

in impaired membrane transport mechanisms and increased permeability and rupturing of

membranes. Cross-linking of membrane associated polypeptides may result in the inactivation

of enzymes, receptors and ion channels. Photosensitizers with anionic substituents, such as

- 138 -

sulphonate or carboxyl groups, have been observed to localize preferentially in the cytoplasm

and relocate to the nucleus upon illumination (Patito et al., 2001), whereas lipophilic

photosensitizers functionalized with cationic groups are believed to (preferentially) traverse

the mitochondrial membrane and accumulate in the mitochondrion (Dummin et al., 1997) -

the subcellular organelle widely demonstrated to be a key component in the preferred

(apoptotic) cell death pathway. Exactly which physicochemical/structural properties and

mechanisms are behind these specific distributions and localizations and how to maximize

tumour tissue selectivity over normal tissue accumulation are issues still under investigation.

Figure 43 (from Castano et al., 2005) illustrates some of the cellular and molecular

signalling pathways that have been determined to occur in cells treated with PDT in vitro

(Argarwal et al., 1991; Dahle et al., 1997).

Figure 43: Cellular signalling pathways leading to apoptosis in cells after PDT. ROS generation depending photosensitizers localization (Castano et al., 2005)

- 139 -

3.2. Pharmacokinetics, pharmacodynamic and subsequent adverse effects of photosensitizer: example of Photofrin®

3.2.1 Pharmacokinetic

The pK of porfimer sodium (Photofrin®) has been studied in mice, rats, guinea pigs,

dogs, and also humans. The results of pK investigations are basically very similar and are

always the same for both genders. In general Photofrin® is infused at 2 mg/kg in an outpatient

setting. About 24 to 48 hours later illuminations occur generally by a diffusing fiber or more

rarely by a micro lens (which is unidirectional). Depending on the clinical situation light dose

of 150 J/cm2 (lens) or 200-300 J/cm2 is employed.

- Pharmacology study in in vivo models:

For general conditions in mice, no effect was observed in 25 or 50 mg/kg porfimer

sodium administered intravenously. At 100 mg/kg and 200 mg/kg there were some disorders

observed and beyond at 200 mg/kg all animals died at least within 3 days which is not

surprising because this is within the range of the Lethal Dose 50 (LD50). With regard to the

central nervous system, porfimer sodium administered intravenously at doses of 50 mg/kg and

above resulted in decreased movement, lengthened thiopental-induced sleep, and inhibition of

strychnine–induced convulsions. A transient lowering of body temperature at 50 mg/kg in

rats was observed which returned to normal 3 hours later. Movement of isolated guinea pig

ileum was inhibited by porfimer sodium at 10 �g/ml, a plasma level which may be equivalent

to 5 mg/kg administered intravenously to mice. In heart and circulatory system there was no

effect on blood pressure, pulse rate and electrocardiogram when tested in dogs up to the

highest dose of 16 mg/kg. There was no influence on lung function.

- Example of pK study in human:

Following a 2 mg/kg dose of porfimer sodium to four male cancer patients, the

average peak plasma concentration was 15 ± 3 mcg/ml (microgram/ml), the elimination t1/2

was 250 ± 285 hours, the steady-state volume of distribution was 0.49 ± 0.28 l/kg, and the

total plasma clearance as 0.051 ± 0.035 ml/min/kg. The mean plasma concentration at 48

hours was 2.6 ± 0.4 mcg/ml. The influence of impaired hepatic function on Photofrin®

disposition has not been evaluated. In vitro studies have shown that Photofrin® was covered

- 140 -

by protein from human serum at a very high level (approximately 90%). The binding was

independent of concentration over the concentration range of 20-100 mcg/ml. A course of

therapy consisted in one injection of Photofrin® (2 mg/kg administered as a slow i.v injection

over 3-5 min) followed by up to two nonthermal applications of 630 nm laser light. Doses of

300 J/cm of tumour length were used in oesophageal cancer. Doses of 200 J/cm were used in

endobronchial cancer for both palliation of obstructing cancer and treatment of superficial

lesions. The first application of light occurred 40-50 hours after injection. Debridement of

residua was performed via endoscopy/bronchoscopy 96-120 hours after injection, after which

any residual tumour could be retreated with a second laser light application at the same dose

used for the initial treatment. Additional courses of PDT with Photofrin® were allowed after 1

month, up to a maximum of three courses (FDA report, 2000).

- Some specific pK parameters:

A. Absorption-bioavailability

Intravenous (i.v) or intraperitoneal (i.p) single dose (5 mg/kg) pK in mice indicated a

long half-life for plasma elimination of residual [14C]-radioactivity associated with the

porphyrins in porfimer sodium or with metabolites: triexpontentially decrease with

elimination half-lives of about 0.75 (�), 10 (�) and 220 (�) h, following i.v dosing, and

biexponentially after i.p with elimination half-lives of 4 and 220 hours.

B. Metabolism

Biotransformation is difficult to interpret with complex mixtures such as Photofrin®,

and metabolism studies per se were not conducted with Photofrin®. However in a biliary

excretion study in the rat, the amount of haematoporphyrin (HP) monomer excreted in the bile

within 48 hours after dosing was twice the amount of HP injected in the dosing preparation.

This indicates that some of ester/ether linkages in the porphyrin excreted dimer/oligomer

fraction were hydrolysed in vivo.

C. Excretion

There is only one investigation for the elimination profile of the 14C porfimer. Urine

and feces of rats were collected each 24 hours for 7 days. The major route (42 %) of

elimination of 14C porfimer sodium was via the feces, whereas only 4% of the dose was

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excreted in the urine. It is suggested that the fecal route is mainly via bile excretion because

23 % of porfimer were excreted into bile within 48 hours in rats. Also this is in accordance

with human data where excretion data have shown that the major route of elimination is fecal

suggesting biliary excretion of 28 % of the i.v dose over 72 hours.

D. Others

In an animal model (hamster) bearing a pancreatic tumour it was shown that porfimer

had a high affinity to tumour tissue and was retained for a long time in this tumour tissue. In

other similar studies in mice it was shown that the amount of labelled hematoporphyrin

derivate in tumour tissue was in fact higher than in muscle and skin but lower than in liver,

spleen and kidney which were the favoured organs for distribution. The placental transfer of

Photofrin® was studied in pregnant rats (n=5) following a single 20 mg/kg i.v. dose of

Photofrin® given on day 18 of gestation. No porphyrin derivatives were detected in amniotic

fluid or foetuses. The transfer of porfimer sodium into breast milk was studied in lactating rats

(n=5) following a single 20 mg/kg i.v. dose on day 9 after delivery. Trace concentrations of

porphyrins excreted were found in breast milk between 6 and 48 hours after dosing with a

maximum concentration of 7.4 �g/ml at 24 hours post injection (EMEA, 2004).

3.2.2. Pharmacodynamic

A. Distribution in in vivo models

In mice and rats, the results showed that, although most of an i.v. dose was removed

from circulation by the liver, spleen and kidney, potentially useful concentrations were

retained for long periods in other tissues, including implanted human tumours. The protein

binding in rat, dog and human was comparable within the species between 80 and 90 % and

was not dependent on the concentration of porfimer sodium. It could be shown that porfimer

sodium has a strong affinity to lipoprotein, especially to low LDL. In plasma, lipoproteins

may be porphyrin carriers to the tumour tissue and its corresponding receptors.

A specific biodistribution study showed the highest content of Photofrin® in liver,

kidney and spleen (Figure 44). Porphyrin content in tumour is smaller than in these organs,

and the porphyrin concentration does not exceed 10 µg/g wet tissues, while in liver, kidney

and spleen it reaches 70, 20.4 and 20.5 µg/g respectively. Quantification of the distribution

and the pK of Photofrin® in normal and tumour tissue biopsies of the human bile duct have

been investigated. Results demonstrated that Photofrin® accumulated in human bile duct

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adenocarcinoma with a tumour to normal tissue fluorescence ratio of about 2:l after 24 and 48

hours of Photofrin® administration. Skin biodistribution was very low compared with others

organs (Pahernik et al., 1998).

Figure 44 : Example of in vivo Photofrin� biodistribution study: single i.v. administration (20 mg/kg) after 1, 2, 6 and 24 hours in nude mice implanted with a small cell lung carcinoma (Tronconi et al.,

1995)

3.2.3. Side effects

Photofrin® causes long lasting cutaneous photosensitivity, as it is absorbed by the

skin. For this reason, patients who have been treated with Photofrin® need to avoid sunlight

for 4-6 weeks (Dolmans et al., 2003). These side effects are very special and incapacitating.

In fact, all patients who receive Photofrin® will be photosensitive and must observe

precautions to avoid exposure of skin and eyes to direct sunlight or bright indoor light (from

examination lamps, including dental lamps, operating room lamps, unshaded light bulbs at

close proximity, etc.). The photosensitivity is due to residual drug which will be present in all

parts of the skin. Exposure of the skin to ambient indoor light is, however, beneficial because

the remaining drug will be inactivated gradually and safely through a photobleaching reaction.

Many changes at the molecular level have been associated with Photofrin®.

Diminution of the activity of a number of enzymes involved in membrane biosynthesis,

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particularly acyltransferases was noted. Mitochondria are a significant intracellular target for

Photofrin®, and inhibition of the associated respiratory processes produces cell death. Human

bladder transitional carcinoma cells, following sublethal PDT treatment, were found to release

arachidonic acid metabolites, mainly thromboxane B2 and PG E2 in a biphasic manner.

Photocytotoxicity can be partially mitigated by agents such as indomethacin which block the

action of PGE, suggesting a relationship between PGE and photodynamic cell damage. PDT

treatment of macrophages in vitro stimulates the production of tumour necrosis factor (TNF),

a cytokine known to induce haemorrhagic necrosis of tumours. The urine of bladder cancer

patients following Photofrin® has been shown to contain elevated levels of the cytokines

interleukin-1 (IL-1), interleukin-2 (IL-2) and TNF. It is not known whether this is a direct or

indirect effect of PDT. Mitomycin C, which blocks cells at G2/M, enhances the effects of

PDT. In a human colon adenocarcinoma cell line mitomycin C treated cells were shown to

take up significantly higher levels of Photofrin® than untreated controls. Etanidazole,

misonidazole and trifluoro-misonidazole were all found to be photoprotective against

Photofrin® when cells were incubated for 24 hours with Photofrin® under aerobic and limited

oxygen (0.3%) conditions (EMEA, 2004).

4. Nanocarriers players: a way to enhance selectivity and specificity of

photosensitizer to improve their tumour bioavailability

Work has recently focused on designing systems to induce greater selectivity and

specificity of photosensitizer in order to enhance tumour cell uptake. The principle of using

carriers may increase the tumour concentration and thus, lead to high bioavailability to

improve the therapeutic effect.

4.1. Silica-based nanocarriers of photosensitizers: literature survey

Regarding the literature, several ways of nanocarrier’s synthesis have been reported.

Very complex to very simple synthesis has been addressed and products tested both in vitro

and in vivo models.

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-Nanocarrier: photosensitizer covently linked

Ohulchanskyy et al., 2007 report a new formulation of nanoparticles (organically

modified silica (ORMOSIL) nanoparticles) in which the photosensitizer based on

iodobenzylpyropheophorbide, derivated of HPPH, was covalently attached to the silica

matrix. They found that the covalently incorporated photosensitizer molecules retained their

spectroscopic and functional properties and could robustly generate cytotoxic singlet oxygen

molecules upon photoirradiation. The advantage offered by this covalently linked

nanofabrication (20 nm) is that the drug is not released during systemic circulation, which is,

according to the authors, often a problem with physical encapsulation. These nanoparticles are

also avidly uptaken by tumour cells in vitro and demonstrate phototoxic action, thereby

highlighting their potential in diagnosis and PDT of cancer.

Brevet et al., 2009 report new class of mesoporous silica nanoparticles based on

Si(OEt)4±mannose with size approximately of 100 nm. Particles were elaborated by covalent

incorporation of a water-soluble photosensitizer and by covering their external surface with

mannose residues. Authors have proved that these mannose functionalized mesoporous silica

nanoparticles presented a much higher in vitro photoefficiency (MTT test) in MDA-MB-231

breast cancer cells than non-functionalized nanoparticles. The higher efficiency of mannose-

functionalized mesoporous silica nanoparticles must be due to an active endocytosis via

mannose receptors.

Rossi et al., 2008, in a chemical study, present the synthesis of Pp IX silica

nanoparticles. The entrapment of Pp IX in silica spheres was achieved by modification of Pp

IX molecules with an organosilane reagent. Pp IX was firmly attached to the silica matrix.

Generation of singlet oxygen was measured in higher proportion than free photosensitizer.

This means that the immobilization of the Pp IX molecules increased the potential of the

photosensitizer to perform PDT.

Tu and colleagues, 2009 report a surface modification process that conjugates a

photosensitizer, Pp IX, with mesoporous silica nanoparticles through covalent bonding to

obtain Pp IX-modified nanoparticles for PDT study. Mesoporous nanoparticles were

hexagonal disk shape with average particle size of 110 nm and thickness of 90 nm. In vitro

tests performed with HeLa cells revealed high cellular-uptake efficiency and the phototoxicity

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was found to be both irradiation time- and dosage-dependent. In order to determine the proper

incubation time, HeLa cells were incubated with nanoparticles suspension for 0.5, 1, 2, and 4

hours. Authors found that after 2 hours of incubation, most cells (95%) have uptaken

nanoparticles. Cells treated with nanoparticles showed significant intracellular staining due to

Pp IX in the cytoplasm, indicating the accumulation of nanoparticles. The cytotoxicity

performed after 2 hours treatments were examined using the MTT assay. Results indicated

that both necrosis and apoptosis can be induced in HeLa cells after nanoparticles-mediated

PDT.

- Nanocarrier: photosensitizer no covently linked

Roy, and colleagues, (2003) report encapsulation of the hydrophobic photosensitizer

HPPH, which is currently undergoing phase I and II clinical trials for esophageal cancer, by

controlled hydrolysis of triethoxyvinylsilane in micellar media. In vitro experiments showed

significant level of cell death for both HPPH-Tween-80 micelles and HPPH-nanoparticles (30

nm) on HeLa and UCI-107 ovarian carcinoma cell lines.

Yan et al., 2003 compare the spectroscopic properties of free meta-

tetra(hydroxyphenyl)-chlorin (m-THPC) and of m-THPC embedded in silicon-based

nanoparticles. An interesting result was that 1O2 delivery by the nanoparticles exceeded that

of the free m-THPC.

Tang et al., 2005 show study of nanoparticles loaded with methylene blue (MB).

Encapsulation of MB was performed in three types of sub-200 nm nanoparticles:

polyacrylamide, sol-gel silica and organically modified silicate (ORMOSIL). In vitro PDT

study using the MB-loaded polyacrylamide nanoparticles was conducted on rat C6 glioma

tumour cells with positive photodynamic results. The 3 silica-based nanoparticles are worth

undergoing additional investigation.

He et al., 2009 have developed bifunctional nanoparticles-based carrier for

simultaneous PDT and in vivo imaging by encapsulating MB alone in a phosphonate

terminated silica matrix. Both in vitro and in vivo PDT studies were performed on HeLa cells

treated with MB-encapsulated silica nanoparticles. In vitro cytotoxicity (without activation)

tests on HeLa cells were performed. Concentration of the MB-encapsulated nanoparticles

with 1 mg/ml was the optimal concentration for efficient phototoxicity with minimum

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cytotoxicity without activation. Then, in vivo tests were investigated on mice bearing Hela

tumours and preliminary results showed that particles can effectively induce cell death.

Furthermore, it was possible to visualize in vivo in real time tumour obtained with optical

imaging system.

4.2. Selection the of simplest process route to design Pp IX silica-based nanocarrier

“One Pot Synthesis of New Hybrid Versatile Nanocarrier Exhibiting Efficient Stability in Biological Environment for U se in Photodynamic Therapy”

Edouard Thiénot, Matthieu Germain, Kelthoum Piejos, Virginie Simon, Audrey Darmon, Julie Marill, Elsa Borghi, Laurent Levy, Jean-François Hochepied, Agnès Pottier ; Submitted

At present, it is commonly admitted that the ideal photosensitizer would be a

chemically stable and pure drug with preferential uptake in tumour, rapid clearance, and a

strong absorption peak at light wavelengths > 630 nm. Above all, low toxicity and high

selectivity are the primary characteristics highlighted by clinicians.

This article describes the chemical possibilities to define the optimal components and

the simplest structure of these products for intended use in the clinics. This paper underlines

the key synthesis parameters that may tune the size of the silica-based nanocarriers within the

nanometer range, the stability studies in mouse serum media, and the interest to add a second

biocompatible inorganic coating to tune the flexibility, hence the permeability, of the silica

core and to improve the stability of the silica-based protoporphyrin IX (Pp IX) nanocarriers.

For additional results, in vitro cell viability tests have been engaged in order to confirm the

biological activity of nanocarriers in our cellular model.

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ARTICLE -II-

One Pot Synthesis of New Hybrid Versatile Nanocarrier

Exhibiting Efficient Stability in Biological Enviro nment

for Use in Photodynamic Therapy

Edouard Thiénot, Matthieu Germain, Kelthoum Piejos, Virginie Simon, Audrey

Darmon, Julie Marill, Elsa Borghi, Laurent Levy, Jean-François Hochepied,

Agnès Pottier

(Submitted)

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4.3. Discussion

Different nanoparticle chemistries have been developed in the nanoPDT platform to

define the optimal components and the simplest structure of these products for intended use in

the clinics. Further, most efforts have been focused on selecting one product which low

complexity avoids as much as possible metabolic and tissue interactions and fulfills the

following conditions. First to keep the simplest synthesis route, avoiding the use of targeting

agent but taking advantage of the EPR effect that tumour tissues offer, owing to their “leaky”

vasculature. Then, to keep stability such as ability to produce ROS of the encapsulated

photosensitizers, at least for the necessary time to accumulate within tumour tissues and to

produce its effect upon light activation.

At the first step, synthesis of monolayer Pp IX silica nanoparticles was detailed for

different sizes. Nanoparticles are spherical and highly monodisperse with sizes of 10±5 nm,

25±5 nm, 65±20 nm, 90±20 nm and 160±30 nm. In fact, initial micelle formation is affected

by various factors including temperature, ionic strength, pH, surfactant species etc. For non-

ionic surfactants, the critical micelle concentration (CMC) decreases with an increasing

temperature due to an increase in hydrophobicity (Kim et al., 2004). Balance between

hydrophobicity and hydrophilicity may change and induce increase of micelle size. For

control of the size of the Pp IX silica-based nanocarriers from 10 nm to 200 nm, temperature

was increase from 18°C to 37°C.

With the ambition to optimize the in vivo performance of the nanoPDT, stability

studies of the Pp IX silica-base nanocarrier in biological media were developed. Pp IX has

specific absorbance and fluorescence spectra which permitted to simplify the stability studies

in mouse serum media. Results showed that Pp IX silica-based nanoparticles present a

modification of Pp IX absorption spectrum after t=2 hours aging in 100% mouse serum and a

corresponding loss of Pp IX fluorescence emission.

In order to generate silica-based Pp IX nanocarriers with efficient stabilization in

biological environments, second generation of photosensitizer vehicle was developed. The

hypothesis of adding a second biocompatible coating to tune the flexibility of the silica-based

nanocarrier backbone has been successful. Different sort of coatings such as dextran, silane

PEG (PEG-Si) or aminopropylsilane (APS) and sodium trimetaphosphate (STMP) were

produced and were tested in in vitro viability assays (data not shown) .

The cellular stability assay is based on the preservation of activity of Pp IX following

Pp IX nanocarriers incubation in 100 % fetal calf serum (FCS).

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Dextran is a coating agent commonly used to functionalize oxide nanoparticles, in

particular magnetite nanoparticles contrast agent, to increase their blood circulation. Dextran

is electrically neutral at neutral pH and the binding at the surface of iron oxide is thought to be

labile (physisorption). It is also reported that dextran can be desorbed in biological

environment, yielding to a direct contact between magnetite nanoparticles and biological

compounds (Auffan et al., 2009). Our results show that dextran molecule, added as second

biocompatible coating, did not improve nanoPDT stability in biological media.

Functional silanes develop covalent links with silanol groups present on silica

surfaces. However, in aqueous media and particularly under neutral pH conditions,

polymerisation of silanes competes with silane-silica surface anchoring (De Monredon et al.,

2004). Hence, heterogneous silane deposition on nanoPDT surface is likely. Indeed, we did

not observe improved nanoPDT stability in biological media using functional silane as second

biocompatible coating agent.

Phosphate compounds, such as sodium trimetaphosphate are considered as complexing

agent for most oxide surface. However, silica surface behaves differently. Furthermore, both

silica surface and sodium trimetaphosphate are negatively charged at neutral pH. We

hypothesized that sodium cations, present in the solution, drive the interaction between

phosphate compound and nanoPDT silanol surface groups. Indeed, Pp IX silica-based

nanoparticles coated with STMP, the bilayer nanocarriers, showed the best stability in

biological media, highlighting the importance of a second specific biocompatible coating.

Roach et al., 2005 report protein adsorption (BSA and fibrinogen) onto model

hydrophobic (CH3) and hydrophilic (OH) surfaces. Authors investigated proteins adsorption

using quartz crystal microbalance (QCM) and grazing angle infrared spectroscopy. The data

show that albumin undergoes adsorption via a single step whereas fibrinogen adsorption is a

more complex, multistage process. Albumin has a stronger affinity toward the CH3 compared

to OH terminated surface. In contrast fibrinogen adheres more rapidly to both surfaces, having

a slightly higher affinity toward the hydrophobic surface. Conformational assessment of the

adsorbed proteins shows that after initial 1 hour incubation few further time-dependent

changes are observed. Both proteins exhibited a less secondary structure upon adsorption onto

a hydrophobic surface than onto a hydrophilic surface, with the effect observed greatest for

albumin. Data presented in this article suggest that proteins (albumin and fibrinogen) are able

to interact with hydrophilic surface with a stronger affinity with hydrophobic surface. Their

adsorption may further induce a conformational change in protein structure. In our present

study, interaction of silica-based nanocarriers and protein from serum is likely to occur.

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Albumin is the most abundant protein in serum and it is suggested that its interaction with

hydroxyl group of silica-based nanocarriers readily occurs. However, the 3 dimensional

structure of the nanocarrier backbone present some flexibility and hydrophobic vinyl groups,

initially constraint within the core of the nanocarrier upon exposure to aqueous environment,

may get oriented toward the external surface, triggered by albumin, upon exposure with serum

media. Such affinity - vinyl hydrophobic groups / albumin - could be an explanation for the

loss of stability of the entrapped Pp IX which may become exposed to the serum media.

Interestingly, 50᧡ effective concentration (EC50) value of Pp IX silica-based nanocarriers,

the monolayer nanocarrier, incubated in 100% FCS media, decreases with incubation time

and reaches the EC50 value of free Pp IX in serum media. The role of STMP would then to

stiffen the silicone backbone of the silica-based nanocarrier by forcing the silanol groups to

interact with the phosphate groups, likely via sodium counter ions. Hence, confining the

hydrophobic vinyl groups within the core of the nanocarrier and stabilizing the Pp IX

molecules toward external media.

The ability of STMP modified Pp IX silica-based nanocarrier, the bilayer nanocarrier,

to kill cells, seems to be preserved for enough time to accumulate within tumour tissues and to

produce its effect upon light activation. Indeed, for the highest STMP content, bilayer

nanocarriers in vitro data, show preservation of Pp IX activity up to 12 hours. The second

generation of nanoPDT products, the bilayer nanocarriers, anticipate promises for efficient in

vivo efficacy, due to their enhance stability – ability of the entrapped photosensitizers to

generate ROS upon laser light excitation – in biological environment.

5. Interaction of Pp IX silica nanoparticles with biological systems

5.1. “Pp IX Silica Nanoparticles Demonstrate Differential Interactions with In Vitro Tumour cell Lines and In Vivo Mouse Models of Human Cancers”

Virginie Simon, Corinne Devaux, Audrey Darmon, Thibault Donnet, Edouard Thiénot, Matthieu Germain, Jérôme Honnorat, Alex Duval, Agnès Pottier, Elsa Borghi, Laurent Levy, Julie Marill ; Photochemistry, Photobiology, 2009

In the recent years, PDT has been successfully used in various cancers: skin, lung,

uterus, gastrointestinal. Nevertheless, because of important side effects such as long lasting

skin phototoxicity, PDT clinical application has been narrow. One of the most potential

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revolution in cancer care was the use of nanoparticles. As shown in first paper, Nanobiotix

created and developed a new alternative for the therapy photodynamic: the nanocarriers. A

drug, Pp IX has been chosen to be encapsulated in a porous silica shell.

This second paper describes the in vitro and in vivo studies performed with Pp IX

silica nanoparticles, the monolayer nanocarrier. Different parameters have been studied to

obtain the best nanoparticles efficacy such as incubation time, activation time, particles

concentrations, post-illumination time, with the weaker nanoparticles toxicity. Preliminary

tests permitted to compare nanoparticles with free Pp IX, the monomer of Photofrin®

sensitizer. Furthermore, efficacy was compared on various tumour cells lines, adherent (HCT

116, HT-29, epidermoid cell line A431, MDA-MB 231 and MCF7) and in lymphoblastoid

suspension cell line (LLBC37).

In this work, in vivo studies were performed in tumour bearing animals –2 human

epithelial cancers (HCT 116, A549) and 1 human Glioblastoma Multiforme – which permitted

first observations on tumour ⁄ skin ratio accumulation of the product. Concerning time

accumulation dynamic, tumour cell type is likely a major determinant but tumour

microenvironment could more influence the differential observed.

In this work, Pp IX was successfully encapsulated into a silica nanoparticle that

preserves the photodynamic activity of the photosensitizer. Our results demonstrate that the

encapsulation in nanoparticles greatly improves the photo-stability and photodynamic efficacy

of Pp IX. Furthermore, it opens some fields for exploration regarding the differential

biodistribution of the nanoparticles in tumour models with various origins, and xenografted

using two different approaches, human tumour cell lines and fragment of tumour from a

patient. Stroma, vessels irrigating the tumour, and even growth conditions may trigger cell

adaptative functions and cytokine secretion, which support tissue and cell differential

biodistribution. Next step of research is to be focused on in vivo efficacy of epithelial and

non-epithelial models.

These findings suggest that Pp IX and probably other photosensitizers encapsulated

could be a promising approach not only for improving PDT efficacy but rather to also explore

other route of administration, and thus trigger a more specific based-disease of PDT in the

clinical setting.

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ARTICLE -III-

Pp IX Silica Nanoparticles Demonstrate Differential

Interactions with In Vitro Tumour cell Lines and In Vivo

Mouse Models of Human Cancers

Virginie Simon, Corinne Devaux, Audrey Darmon, Thibault Donnet, Edouard

Thiénot, Matthieu Germain, Jérôme Honnorat, Alex Duval, Agnès Pottier, Elsa

Borghi, Laurent Levy, Julie Marill

(Photochemistry, Photobiology, 2009)

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5.2. Discussion

In vitro tests investigation represent a sophisticated and reproducible system with

which basic questions can be answered and which may help to understand what happens in

vivo. Cell culture experiments allow for comprehensive investigations of particles-cell

interactions. These experiments are necessary to study the effects of Pp IX silica nanoparticles

on intracellular uptake, transport and subcellular nanoparticles localization.

This article introduces some advanced researches in in vitro and in in vivo models with

the ambition to improve knowledge on the role of biological factors in the photodamage.

At first toxicity and efficacy of Pp IX silica nanoparticles have been investigated. The WST-

1 cell proliferation assay has been chosen. It is a colorimetric assay which is based on the

cleavage of a tetrazolium salt, 3-(4,5-diMethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-

(4-sulfophenyl)-2H-tetrazolium inner salt (MTS), by mitochondrial dehydrogenases to form

formazan in viable cells. WST-1 has many advantages: it can be used to assay either adherent

or suspension cell, it is very sensitive, it can detect 500 to 50.000 cells in a single well of a 96-

well plate and it is possible to test of lot of conditions (concentrations, different products,

controls…) in only 1 experiment. 3 hours of incubation and 20 min of illumination has been

defined as the best particles efficacy conditions with an EC50 for 6 different cells lines

(adherent or suspension cells) between 0.40 µM ± 0.01 for the LLBC37 and 1.13 µM ± 0.15

for the MDA-MB-231. In these conditions, Pp IX silica nanoparticles were always more

efficient (more than 1 Log) than free Pp IX.

Pp IX silica nanoparticles are able to enter into all cells lines tested and diffused in all

cytoplasm. The mechanism by which nanoparticles penetrate in HCT 116 cells without

specific receptors on their outer surface is assumed to involve a passive uptake. In fact, cell

viability assays performed at 4°C showed EC50=0.4 µM (same than at 37°C). Others

molecules such as glucose or homeoproteins (Holcman et al., 2007) diffuse through cellular

membranes by passive penetration. Pp IX nanoparticles passive uptake may be initiated by

Van der Waals forces, electrostatic charges, steric interactions, or interfacial tension effect.

Others tests should be investigated in order to determine the passive way (for instance

spectroscopy correlated with fluorescence) and extended to other cells types. Chithrani et al.,

2007 showed gold size effect for nanoparticle uptake (receptor-mediated endocytosis) and

clearance (the smaller exocytosed more rapidly). In this in vitro study, no size effect (10 to 60

nm) on HCT 116 uptake and cell viability was showed (EC50=0.4 µM). It was no surprising to

quantify the same total amount of particles whatever the size considering the role of passive

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uptake. In fact, in the passive diffusion process, the membrane behaves as an inert lipid–pore

boundary, and nanoparticles traverse this barrier either by diffusion through the lipoprotein

region or, alternatively, filtering through aqueous pores (channels) without the cellular

expenditure of energy (Mottier at al., 2006). In addition, for the first time, clearance of silica

nanoparticles on tumour cells is established. Only 24 hours after the end of incubation all Pp

IX silica nanoparticles were cleared. However, silica shells trafficking at cellular level was

different than metallic particles such as gold (Chithrani et al., 2007) as no size effect on

nanoparticle trafficking is observed (passive way is thought to participate to cellular uptake).

Pp IX silica nanoparticles total clearance in HCT 116, 24 hours after medium renewal,

suggested that we have to work on the therapeutic window for in vivo application in order to

optimize schedule for nanoparticles activation.

In vivo studies were performed in tumour bearing animals which permitted first

observations of nanoparticles distribution and tumour/skin ratio accumulation. The 3 tumour

models behaved differently according to the maximal accumulation time point: 20, 16 and 2

hours for HCT 116, A549 and for Glioblastoma Multiforme respectively. Tumour and liver

was the target in terms of nanoparticle-related fluorescence. These results proved that tumour

cell types are likely a major determinant in biodistribution. Furthermore, tumour environment

such as stroma could more influence this differential time of accumulation dynamic

(including nanoparticles internalization and clearance). The ECM could act as a dispersive

filter, controlling the composition of extracellular fluid and the rate of molecular trafficking

(Muerkoster et al., 2008; Jackson et al., 2008; Minchinton et al., 2006; McKee et al., 2006;

Pluen et al., 2001 and Netti et al., 2000).

Finally, deeper comprehension of cell death mechanism was detailed in this article.

ROS generation has very short migration distance and was colocalized with nanoparticles

after activation. Nanoparticles localization was key of the efficacy. Higher particles

concentrations yielded more marked difference and higher ROS amounts caused larger cell

photodamage and consequently larger cell death.

We have successfully controled Pp IX silica nanoparticles parameters to optimize the

in vitro efficacy. First in vivo biodistributions were very encouraging because of the specific

tumour accumulation in 3 different models, especially with Glioblastoma Multiforme model.

Glioblastoma Multiforme is the most aggressive of the primary brain tumours and could be

chosen as cancer model for clinical study. In vivo efficacy should be investigated and

nanoparticles synthesis improved to follow the preclinical steps advancement for medical

device development.

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6. Generals PDT conclusions and perspectives

PDT has emerged as one of the important therapeutic options in management of cancer

(Allison et al., 2008). PDT leads to selective and irreversible destruction of diseased tissues,

without damaging adjacent healthy ones. Despite its advantages over current treatments, PDT

is yet to gain general clinical acceptance. There are several technical difficulties in the

application of PDT to a wide range of diseases. First currently FDA approved PDT

photosensitizers, absorb in the visible spectral region below 700 nm, where light penetration

into the skin is only a few millimeters, thus clinically limiting PDT to treat surface and

relatively superficial lesions. However, this has to be weighed against the availability of

advanced fibre-optic scopes which can reach most body cavities. Second is the difficulty in

preparing pharmaceutical formulations that enable parenteral administration because most

existing photosensitizers are hydrophobic and aggregate easily under physiological condition.

Thirdly, the accumulation selectivity to diseased tissues is often not high enough for clinical

use. Regarding all PDT limitations, we tried to define the best photosensitizer nanocarriers.

What is the ideal system for the next generation?

Regarding the literature, MacRobert exposed “the ideal photoproperties for a

sensitizer” (MacRobert et al., 1989):

� be chemically pure, at least have a known structure, or consist of a well defined

mixture.

� have a minimal dark toxicity and only become cytotoxic in presence of light.

� be preferentially retained by the target tissue (specificity),

� be rapidly excreted from the body (no systemic or skin photosensitizing effects)

� have a high photochemical reactivity (high triplet state field, long triplet state

lifetimes)

� be able to effectively produce singlet oxygen and other reactive oxygen species

� have a strong absorbance with a high extinction coefficient for the wavelength range

600-800 nm, where tissue penetration of light is high.

All these criteria were examined and tested on both chemistry and biology research

team. Nanoparticles offer solutions to each of the three difficulties. Nanoparticles carrying

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photosensitizer by different strategies (in particular photosensitizer encapsulation) offer

benefits of hydrophilicity and appropriate size for passive targeting to tumour tissues by the

enhanced EPR effect. Selective accumulation can be enhanced by modifying the monocarrier

surface using targeting agent such as mAb or specific tumour seeking molecules. Pp IX silica

nanoparticles has a lot of advantages: very simple chemistry, better efficacy than free Pp IX

for all in vitro cells lines tested, good tumour biodistribution. Further, improved stability in

biological media is expected with second generation of nanoPDT products, the bilayer

nanocarriers.

Efficient PDT may increase the choice of anticancer modalities, opening the

possibility of a “new line of local treatment”. Pp IX silica nanoparticles hold promises to

enhance the current therapeutic window.

What kind of tests for the future?

For the future, it will be interesting to test biodistribution of Pp IX-STMP coated

nanoparticles, the bilayer nanocarriers. Nanocarriers with bilayer coating could have different

time of tumour maximal accumulation and potential others organs target. For Pp IX silica

nanoparticles application it is necessary to decrease side effect by comparison with

Photofrin®. As we know, Photofrin® has significant long lasting skin accumulation and it will

be necessary to quantify the nanoPDT distribution in this large organ. Deeper exploration of

pK characteristics on skin, tumour and liver should be planned.

In all in vitro and in vivo tests 25 nm Pp IX silica nanoparticles were studied. In HCT

116 viability and quantification tests showed that there are no size effect between 10-60 nm

ranges. Approximately 0.7 molecule of Pp IX per particle could be encapsulated in 25 nm

silica shell. Hence, 60 nm silica nanoparticles should allow for a much higher photosensitizer

encapsulation. If we increase the total amount of Pp IX per particle it will be theoretically

possible to increase efficacy. If chemical department research succeeds to encapsulate 7

molecules of Pp IX per particles (ten times higher) it will be interesting to perform

biodistribution studies with monolayer and bilayer nanocarriers with size of 60 nm. Then, if

particles accumulate into the tumour, further in vivo efficacy tests should be run.

Finally, evaluation of subcellular localisation in healthy and human tumour cell lines

could be very useful for the complete in vitro nanocarrier comprehension.

Such hybrid versatile nanocarrier is expected to reach one of the key challenges of the

domain by allowing the delivery of the nanocarriers at the right site and the right dose.

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GENERALS CONCLUSIONS AND

PERSPECTIVES

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The scope of this PhD work is to broaden the current thinking and knowledge in view

of recent developments in relation to nanotechnology-based oncology products. Specifically,

study of interactions between nanoparticles activated by external electromagnetic energy

sources and cancer cells for enhancement of therapeutic window was performed.

Nanotechnology is able to design nanomaterials with new modalities such as the use

of active products for breakthrough in cancer care. Nanotechnology may also bring an

enabling function such as the use of nanocarriers for impacting the pK of molecules and

introducing the possibility of controlling sub-cellular bioavailability. Nanomaterials size

allows interactions with biological entities, hence allows pathways to interact with biology.

However, communication has to be established and required a deep appreciation of the

nanoparticles and biological entities interaction for safe and efficient use of nanoparticles in

biology. When nanoparticles meet medicine, new possibilities for cancer treatment are

envisaged such as the use of outside body energy source to innovative products activation.

This PhD work participates to the development of two types of nanoparticles,

NBTXR3 and nanoPDT, intended with different rational for design. For both nanoparticles,

study of interactions between nanoparticles and cancer cells was performed. NBTXR3

activated with typical radiotherapy treatments or nanoPDT nanocarriers developed for PDT

have clearly for ambition to enlarge the therapeutic window. Both therapeutic products are

intended to make the cancer treatment more effective, less deadly to healthy tissues.

NBTXR3 is the lead product of nanoXrayTM platform. We have demonstrated on

colon cancer cell lines that NBTXR3 nanoparticles penetrate cells via endocytosis ; that the

NBTXR3 nanoparticules are into the cytoplasm into endosomes and then lysosomes; showed

the importance of NBTXR3 nanoparticles cell internalization to achieve a significant higher

radiosensitization effect; suggested that NBTXR3 nanoparticles appear to have long residence

within cells; visualized that endosomes containing NBTXR3 nanoparticles fused into the

cytoplasm and that NBTXR3 nanoparticles activated by ionizing radiation increase

micronuclei formation. Finally, the highest efficacy appears to be correlated to the maximum

amount of nanoparticle per cell.

Results form the basis for the understanding of factors, which are determinant of the

significant radiation enhancement demonstrated by irradiated NBTXR3 nanoparticles on

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colon cancer line models. Moreover they have yield foundations to establish schedule of

NBTXR3 nanoparticles administration in in vivo models and the modality of ionizing

radiation delivery. Currently, preclinical studies are ongoing. Preliminary in vivo efficacy

study was conducted in mice bearing HCT 116 tumours grafted on the flank: NBTXR3

nanoparticles in combination with radiotherapy leads to a total regression of tumour on all

animals compared to control (treated mice subjected to radiotherapy alone) (data not shown).

Among the next steps, the use of different types of energy and deeper studies of

biologic specificity are important research axis which should be addressed. Indeed, many in

vitro studies should be completed such as define the type and proportion of cell death which is

induced by the nanoparticles for both tumour and healthy cell lines. Then, we should prove

that NBTXR3 is the ideal nanoparticle according to its size and surface coating in order to

optimize biodistribution and/or cellular uptake and/or localization at the subcellular level. We

should then evaluate their efficacy and toxicity in vivo in other tumours cells lines than HCT

116.

Regarding these results, NBTXR3 nanoparticles offer a breakthrough approach to

create efficient pathways to cancer therapy. NanoXrayTM plateform is a new treatment

weapon that could be used alone, or in concert with existing anticancer protocols:

chemotherapy, surgery, targeted molecules and immunotherapy. Efficacy is expected to be

proportional to the duration of activation and the number of radiotherapy sessions.

NanoPDT plateform develops photosensitizer nanocarriers. We have proved that

silica-based nanocarriers are efficient carriers for drugs to protect the drug from exposure to

aqueous environment; that silica-based nanocarriers present a relevant range of porosity in

aqueous solution to efficiently entrap the Pp IX photosensitizer (a physical encapsulation of

Pp IX molecule within nanocarriers was performed with the will to simplify as much as

possible both the product synthesis route and its final composition); that the addition of a

second coating to form a bilayer monocarrier significantly preserves the ability of the Pp IX

silica-based nanocarriers to kill cell after aging in 100% FCS media.

In addition, previous preclinical studies have demonstrated in vitro the essential non

toxicity of this Pp IX silica nanoparticle formulation and interestingly, a very good tolerance

in in vivo models. We showed that Pp IX silica nanoparticles of 10-60 nm range did behave in

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the same way concerning cell uptake and cell viability; that internalization mechanism

involves a high proportion of passive internalization with accumulation within the cytoplasm

of cells; that Pp IX silica nanoparticle uptake was dependent upon cell type, as shown by the

greater uptake into HCT 116 than HT 29 cells; that the experiments supposed a saturation

threshold in their intracellular accumulation at this time point without any evidence of toxicity

and that for the first time, clearance of silica nanoparticles on tumor cells was reported. For all

the 6 cell lines tested, nanoparticles localization was diffuse into the cytoplasmic area.

Furthermore, ROS generation was significantly improved in the presence of Pp IX silica

nanoparticles in both HCT 116 and HT-29 cells lines: higher concentrations yielded more

marked difference. Higher ROS amounts caused larger cell photodamages and consequently

better phototoxic effects. For all tumour cells lines tested in vitro, Pp IX silica nanoparticles

were more efficient than free Pp IX.

In vivo studies were performed in tumour-bearing animals which permitted first

observations on tumour ⁄ skin ratio accumulation of the product. Kinetics demonstrated by

semi-quantitative analysis showed that tumour models behaved differently according to the

maximal tumour accumulation time point. Liver fluorescence intensity remained remarkably

constant over the time period investigated and no urine excretion was found.

Recently Nanobiotix has published results of a preclinical study with product issued

from the nanoPDT technology platform: the preclinical study has validated the applicability

of using nanoPDT to treat glioblastoma multiforme, one of the most prevalent brain tumours.

Many in vitro studies should be further addressed such as characterize the

nanoparticles toxicity without activation for healthy cell lines; define the type and proportion

of cell death which is induced by the nanoparticles for both tumour and healthy cell lines;

define the sub-localization of nanoparticles into cells; explore the specific endocytosis

players; precise uptake and clearance ratios between tumour cell lines; test the biodistribution

and the biocompatibility of different size of Pp IX silica nanoparticles as well as the bilayer

nanocarriers and finally test the in vivo efficacy of theses products in various tumour cells

lines such as HCT 116 and glioblastoma multiform models.

Of course, there is much more work to be done and preclinical findings hold promises

for the future development of nanoPDT technology.

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RESUME DETAILLE DE LA THESE

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TABLE DES MATIERES

I-RECHERCHE EN NANOMEDECINE ............................................................................... - 175 -

1.1. LA NANOMEDECINE: QUAND LES NANOTECHNOLOGIES RENCONTRENT LE DOMAINE DE LA

SANTE ................................................................................................................................... - 175 - 1.1.1 L’importance d’être à l’échelle nanométrique pour interagir avec le monde biologique .................................................................................................................. - 175 - 1.1.2 Définition de la nanomédecine ......................................................................... - 175 - 1.1.3 Quels nanomatériaux peuvent jouer un rôle en médecine et pourquoi ............ - 176 -

A- Les nanomatériaux organiques : systèmes de libération contrôlée de molécules thérapeutiques .............................................................................................................. - 176 - B- Les nanomatériaux inorganiques : une nouvelle modalité de traitement .............. - 176 - C- L’avenir des nanomatériaux en nanomédecine ...................................................... - 176 -

1.2. LA MALADIE CANCEREUSE ............................................................................................ - 177 - 1.2.1. Le cancer ......................................................................................................... - 177 -

A- Epidémiologie ........................................................................................................ - 177 - B- Caractéristiques moléculaires du cancer ................................................................ - 177 - C- Principaux traitements pour le cancer ................................................................... - 178 -

1.2.2. Etroitesse de la fenêtre thérapeutique: la limitation majeure des traitements anti-cancéreux ................................................................................................................... - 179 -

A- Les limitations des traitements anti-cancéreux ...................................................... - 179 - B- L’élargissement de la fenêtre thérapeutique ........................................................... - 179 - C- Comment réconcilier la dose tolérée avec la dose curative.................................... - 182 -

II-DES NANOPARTICULES ACTIVABLES POUR LE TRAITEMENT DU CANCER : UN MOYEN D’ELARGIR LA FENETRE THERAPEUTIQUE DES APPROCHES ANTI-CANCEREUSES .................................................................................................................... - 183 -

2.1 INTRODUCTION ............................................................................................................... - 183 - 2.1.1 Nanobiotix développe des nanomatériaux ‘activables’ pour apporter des solutions cliniques aux patients ................................................................................................ - 183 - 2.1.2 Interaction entre les nanomatériaux et le milieu biologique : une compréhension nécessaire pour amener les nanomatériaux en clinique............................................. - 185 - 2.1.3 Présentation du travail de thèse : participation au développement de deux types de nanoparticules pour le traitement du cancer selon deux approches différentes ........ - 186 -

2.2 LA THERAPIE NANOXRAYTM

PERMET UNE AUGMENTATION SIGNIFICATIVE DE LA DOSE DE

RAYONS X AU NIVEAU DE LA TUMEUR SANS CHANGER LA DOSE APPLIQUEE AUX TISSUS SAINS- 190 - 2.2.1 Nanoparticules activables par rayons X : mécanisme d’action de NBTXR3 .. - 190 - 2.2.2 Etat de l’art : des nanoparticules d’or activables par radiation ionisante pour traiter le cancer .................................................................................................................... - 190 -

A- Trafic intracellulaire des nanoparticules d’or ........................................................ - 191 - B- Efficacité in vitro et in vivo des nanoparticules d’or ............................................. - 192 -

2.2.3. Augmentation de l’effet de la radiothérapie par les nanoparticules nanoXrayTM ..... - 194 -

Article 1: « Importance de la biodisponibilité des nanoparticules NBTXR3 dans les endosomes pour optimiser l’efficacité de la radiothérapie sur des cellules tumorales humaines de colon » .................................................................................................... - 194 -

- 174 -

2.3 DES PP IX NANOTRANSPORTEURS CREENT UNE LOCALISATION ET UNE BIODISTRIBUTION

ENTRE LA TUMEUR ET LES TISSUS SAINS DIFFERENCIEES: ELARGISSEMENT DE LA FENETRE

THERAPEUTIQUE DE LA THERAPIE PHOTODYNAMIQUE .......................................................... - 195 - 2.3.1 Mécanisme d’action de la thérapie photodynamique ....................................... - 195 - 2.3.2 Présentation des photosensibilisants avec autorisation de mise sur le marché - 195 - 2.3.3 Administration par voie générale des photosensibilisants destinés à un traitement local ........................................................................................................................... - 196 -

A- Principales caractéristiques pharmacocinétiques du Photofrin® ............................ - 196 - B- Effets secondaires du Photofrin® ............................................................................ - 196 -

2.3.4 Les nanotransporteurs ...................................................................................... - 197 - A- Etat de l’art des nanotransporteurs de silice ........................................................... - 197 - B- Nanobiotix a sélectionné un procédé de synthèse simple pour développer des nanotransporteurs contenant le photosensibilisant Pp IX. .......................................... - 197 - Article 2: « Synthèse d’un nouvel hybride polyvalent (nanotransporteur), montrant une stabilité adaptée dans un environnement biologique pour une utilisation en thérapie photodynamique » ....................................................................................................... - 197 -

2.3.5 Interactions entre les nanoparticules de silice encapsulant le Pp IX et des systèmes biologiques ................................................................................................................ - 198 -

Article 3: « Des nanoparticules de silice encapsulant le photosensibilisant Pp IX présentent des interactions spécifiques avec des lignées cellulaires tumorales in vitro et des modèles de souris porteuses de cancers humains in vivo” ................................... - 198 -

III-RESULTATS ..................................................................................................................... - 199 -

IV-CONCLUSIONS ET PERSPECTIVES GENERALES .................................................... - 204 -

V-REFERENCES BIBLIOGRAPHIQUES ............................................................................ - 206 -

- 175 -

I-RECHERCHE EN NANOMEDECINE

1.1. La Nanomédecine: quand les nanotechnologies rencontrent le domaine

de la santé

1.1.1 L’importance d’être à l’échelle nanométrique pour interagir avec le monde biologique

L’année 1959 est essentielle pour l’essor des nanotechnologies et plus particulièrement

pour son application au domaine de la santé. En effet, Richard Feynman dans son discours

novateur ‘There’s Plenty of Room at the Bottom, An Invitation to Enter a New Field of

Physics”, a été visionnaire en avançant l’idée de construire des objets capables de manœuvrer

à l’échelle des cellules biologiques.

Depuis, les nanotechnologies ont ouvert la voie vers un ‘nanomonde’, et génèrent des

systèmes simples ou élaborés dont la taille varie de quelques nanomètres à quelques centaines

de nanomètres. Ces ‘nanosystèmes’ ont aujourd’hui l’ambition d’occuper une place

fondamentale en médecine. De part leur taille nanométrique, ces ‘nanosystèmes’ ont la

capacité d’interagir avec les entités biologiques telles que les tissus, les cellules, voire

d’opérer au sein même des cellules biologiques.

Cependant, si la taille de ces ‘nanosystèmes’ permet d’agir à l’échelle du monde

biologique, il apparaît essentiel aujourd’hui que ces objets interagissent avec les systèmes

biologiques pour être utilisés sans danger et de façon effective en médecine.

1.1.2 Définition de la nanomédecine

La nanomédecine est l’application de la nanotechnologie au domaine de la santé. La

nanomédecine se réfère à la signification originelle de la nanotechnologie, laquelle se sert des

effets physiques qui se produisent à l’échelle nanométrique dans les objets.

La nanomédecine développe aujourd’hui des systèmes dont les champs d’applications

couvrent les domaines de la vectorisation des médicaments, l’exploration plus ciblée et moins

pénible pour les patients. De plus, un diagnostic plus précoce des maladies peut permettre

d’aboutir à une médecine plus préventive et plus personnalisée, c’est-à-dire prenant en compte

les spécificités biologiques de chaque individu.

- 176 -

1.1.3 Quels nanomatériaux peuvent jouer un rôle en médecine et pourquoi

Les nanomatériaux développés pour des applications en médecine sont fonctions

orientées. Les systèmes de libération de médicaments visent à améliorer le rapport bénéfice

sur risque de certaines molécules thérapeutiques. Ces ‘nanosystèmes’ sont un exemple

typique d’utilisation des nanotechnologies pour apporter une fonctionnalité supplémentaire

bénéfique à la pratique médicale. Les nanomatériaux peuvent aussi, de part leur propriétés

spécifiques, apporter de nouvelles modalités d’usage et permettre de réaliser des avancées

sans précédent pour le traitement des patients.

A- Les nanomatériaux organiques : systèmes de libération contrôlée de

molécules thérapeutiques

Les nanomatériaux organiques englobent une grande variété de structures. Elles ont

comme but commun de transporter et éventuellement de délivrer des molécules au niveau

d’un site spécifique par voie contrôlée. Les applications de nanomatériaux les plus

développées sont à l’heure actuelle celles des liposomes, des micelles, des nanoparticules

polymériques et des dendrimères.

B- Les nanomatériaux inorganiques : une nouvelle modalité de

traitement

Les nanomatériaux inorganiques sont particulièrement prometteurs pour des

applications médicales. En effet, le cœur de certains nanomatériaux possède des propriétés

exclusives à l’échelle nanométrique dues à ce que l’on définit comme l’effet quantique. Le

ratio surface sur volume augmente quand la taille des matériaux décroît et de nouvelles

propriétés, telles que électroniques ou magnétiques, peuvent être observées et exploitées pour

des applications médicales spécifiques. Les applications de nanomatériaux les plus

développées sont à l’heure actuelle celles des nanoparticules superparamagnétiques

(oxydes de fer), des quantum dots et des nanoparticules métalliques (or).

C- L’avenir des nanomatériaux en nanomédecine

Le « National Nanotechnology Initiative » et la « Plateforme Européenne de

Nanomédecine » ont travaillé pour anticiper et définir les futures applications des

- 177 -

nanoparticules dans le domaine de la santé. Les priorités de demain ciblent le diagnostic in

vitro et in vivo, le développement de nanotransporteurs à visée thérapeutique, la recherche de

nouveaux implants et la régénération des tissus, les systèmes biologiques d’ingénierie ainsi

que l’innovation dans le domaine de la médecine instrumentale et des dispositifs médicaux.

1.2. La maladie cancéreuse

1.2.1. Le cancer

A- Epidémiologie

Le cancer demeure l’une des causes majeure de maladie, avec plus de 10 millions de

nouveaux cas diagnostiqués chaque année dans le monde. Dans les pays développés, le cancer

est l’une des principales causes de mortalité : il a tué plus de 6,7 millions de personnes à

travers le monde en 2002. Cette tendance ne va pas être freinée dans les prochaines décennies

et les prévisions restent pessimistes avec une estimation de 10,3 millions de personnes tuées et

plus de 16 millions de nouveaux cas détectés d’ici 2020 (Organisation Mondiale de la Santé ;

OMS, 2007). La figure 1 présente la mortalité à 5 ans pour les cancers les plus répandus :

Figure 1. Statistiques du cancer (Simon et al., 2007)

Les progrès de la médecine et de la recherche pharmaceutique sont importants,

puisque globalement 50 % des cas sont guéris en France ; néanmoins, le traitement contre le

cancer reste plus que jamais un enjeu majeur en terme de santé publique.

B- Caractéristiques moléculaires du cancer

Le cancer ne peut pas être considéré comme une maladie mais comme une multitude

de maladies constituée d’une centaine de sous-types. Les cancers peuvent être classés en deux

catégories : les hématologiques (malignes du sang) et les tumeurs solides. Quand les cellules

du corps deviennent anormales et qu’elles se divisent sans contrôle, la tumeur se forme. Elle

peut être de type maligne ou bénigne. La caractéristique unique, commune à ces cancers est la

25%92%86%44%95%79%27%52%36%54%96%68%Mortalitéà 5 ans

thyroïdeoesophagepoumonprostatefoieestomacseinreinvessiecolorectalpancréasovairesCancers

25%92%86%44%95%79%27%52%36%54%96%68%Mortalitéà 5 ans

thyroïdeoesophagepoumonprostatefoieestomacseinreinvessiecolorectalpancréasovairesCancers

- 178 -

prolifération anormale des cellules. Par des moyens chimiques et mécaniques, les cellules

tumorales vont s’insérer dans l’espace séparant les cellules normales ou à leur niveau, les

tuant. Le cancer est une maladie génétique (Holland et al., 2006). Les bases génétiques de la

tumorigénèse changent énormément d’un cancer à un autre mais les étapes requises pour la

formation de métastases sont similaires pour toutes les tumeurs. Par détachement du site

primitif, les cellules utilisent d’une façon prédominante, soit les vaisseaux lymphatiques, soit

la voie sanguine. Ces étapes impliquent des stimulations lymphangiogéniques et

angiogéniques au niveau de la tumeur mais aussi des perturbations au niveau de

l’environnement local de la tumeur. L’invasion et les métastases tuent leur hôte par deux

processus : l’invasion locale et la colonisation des organes distants et la génération de

dommages cellulaires induits.

C- Principaux traitements pour le cancer

Les trois principaux traitements du cancer sont la chirurgie, la radiothérapie et la

chimiothérapie. Chacun de ces traitements peut être utilisé seul ou en combinaison, selon le

type de tumeur traité. Le choix du traitement va dépendre de la localisation et du type

tumoral, de son stade de développement et de l’état général du patient.

La chirurgie

La plupart du temps, les cancers de type non hématologique peuvent être soignés

intégralement par la chirurgie. La chirurgie est le plus ancien traitement pour le cancer et reste

le traitement d’éradication des cancers solides. Lorsque le cancer est métastasé à d’autres sites

de l’organisme, l’utilisation de la chirurgie devient alors controversée.

La chimiothérapie

La chimiothérapie est un traitement systémique permettant de tuer les cellules

tumorales de localisation primitive et de traiter les métastases éloignées. La chimiothérapie a

énormément amélioré le pronostic de nombreux cancers. Elle reste néanmoins peu curative

exceptée pour quelques tumeurs comme les lymphomes, les leucémies et le cancer des

testicules. Le terme chimiothérapie réfère souvent à des médicaments de type cytotoxiques

qui affectent les divisions cellulaires rapides des cellules anormales. Les médicaments issus

- 179 -

de la chimiothérapie interfèrent à différents stades du cycle de division cellulaire, par exemple

au moment de la duplication de l’acide désoxyribonucléique (ADN) ou à celui de la

séparation des chromosomes néoformés.

La radiothérapie

La radiothérapie est l’utilisation de radiations ionisantes pour tuer les cellules

cancéreuses. Ce traitement va léser ou tuer les cellules tumorales en endommageant le

matériel génétique, les rendant ainsi incapables de continuer de croître et de se diviser. Le but

de la radiothérapie est de créer un maximum de dommages aux cellules tumorales, tout en

affectant le moins possible les cellules saines. Les effets de la radiothérapie sont alors

localisés et confinés à la région du traitement.

1.2.2. Etroitesse de la fenêtre thérapeutique: la limitation majeure des traitements anti-cancéreux

A- Les limitations des traitements anti-cancéreux

Actuellement, les agents anti-tumoraux ciblant le cycle cellulaire et l’ADN, comme les

agents cytotoxiques ou les rayons X, sont les plus efficaces en clinique. Ils ont permis

d’améliorer de manière significative la survie des patients quand ils sont utilisés seuls ou en

combinaison avec d’autres médicaments ayant des mécanismes d’action différents.

Néanmoins, des efforts dans le domaine de la recherche se poursuivent pour améliorer

les traitements actuels. Il s’avère important par exemple de comprendre les voies de

signalisations de transduction qui vont médier les réponses cellulaires. De nombreux espoirs

résident dans la modification du rapport thérapeutique bénéfice sur risque. Malgré les

avancées techniques, la plupart des équipements de radiothérapie actuellement sur le marché

présentent de nombreuses limitations qui restreignent significativement le traitement.

Beaucoup de patients atteints d’une tumeur ne répondent pas aux radiations ou développent

une résistance à celles-ci.

B- L’élargissement de la fenêtre thérapeutique

L’objectif des approches pour traiter le cancer est d’obtenir des traitements présentant

une forte probabilité de guérison avec le minimum de risque pour les tissus sains et une

biodistribution optimale (systémique ou loco-régionale) de l’agent thérapeutique.

- 180 -

Le contrôle du cancer

Le principal obstacle pour traiter de façon effective le cancer est l’échec de la thérapie

initiale. En effet, cette dernière ne permet pas toujours d’éradiquer un nombre suffisant de

cellules tumorales pour éviter la récurrence de la maladie, ceci affectant de façon significative

la survie à long terme des patients. La population de cellules qui a survécu au traitement est

appelée maladie résiduelle microscopique (MRD) (quelques millions de cellules malignes) et

est responsable des rechutes. Ces cellules peuvent trouver refuge dans le microenvironnement,

ou le stroma, qui les protègent.

Les stratégies thérapeutiques sont limitées par le degré de tolérance des tissus sains ;

en effet des doses très élevées permettraient d’éradiquer la totalité des cellules tumorales,

mais entraîneraient également la mort des cellules saines avec des conséquences délétères

pour le patient. Ce problème impose très souvent une limitation pour délivrer la dose

nécessaire au traitement de la tumeur et permet en conséquence à quelques cellules malignes

d’échapper au traitement.

La pharmacocinétique

Le but de la pharmacocinétique est de fournir les connaissances nécessaires à

l'adaptation de la posologie pour obtenir les concentrations plasmatiques suffisantes d'un

médicament entraînant l'effet optimum, c'est-à-dire la meilleure efficacité avec le minimum

d'effets indésirables. En effet, le médicament est inefficace lorsqu’il est administré à de trop

faibles concentrations ; et lorsque celles-ci sont trop élevées, les effets indésirables

prédominent sur l'efficacité.

En pharmacologie clinique, le paramètre facilement et directement accessible est la

concentration plasmatique du médicament. Les différences pharmacocinétiques entre

médicaments proviennent essentiellement de la facilité avec laquelle ils traversent les

membranes biologiques et leur métabolisation par les enzymes présentes chez le patient.

L'acquisition des connaissances pharmacocinétiques est nécessaire à une prescription correcte.

La toxicité

L’un des effets secondaires les plus sévères à une thérapie efficace est le

développement d’un second cancer primitif. Celui-ci reflète non seulement les effets tardifs de

- 181 -

la thérapie, mais aussi le rôle des facteurs environnementaux présents lors de l’apparition du

premier cancer: le tabac, la consommation d’alcool, l’alimentation, la fonction immunitaire, le

statut hormonal et l’exposition environnementale.

Des risques accrus de développement de seconds cancers primitifs sont identifiés après

des traitements par radiothérapie et avec des agents thérapeutiques tels que les agents

alkylants, les inhibiteurs de la topoisomérase et les anti-métabolites.

La fenêtre thérapeutique restreinte

Le choix du traitement par radiothérapie est empirique et essentiellement basé sur la

connaissance du radiothérapeute en fonction de l’organe ciblé. Cent ans de pratique en

radiothérapie n’ont toujours pas permis de définir clairement le régime de fractions optimales

types à appliquer aux patients. La fréquence d’apparitions d’effets secondaires sévères est

dépendante de la dose totale d’irradiation, de la dose par fraction, du temps de traitement

total, du type de rayonnements, de l’énergie utilisée et de la surface totale de la peau exposée

aux rayons. Au fur et à mesure du temps, l’exposition à des radiations au niveau des tissus

sains va entraîner l’accumulation de dommages et limiter la possibilité de futurs traitements.

Il est également admis que la combinaison de la radiothérapie avec d’autres thérapies telles

que la chimiothérapie (chimioradiothérapie) augmente les effets secondaires sévères. Ce

phénomène est particulièrement exacerbé quand cette combinaison se fait avec des protocoles

de fractionnements.

La figure 2B montre une fenêtre thérapeutique restreinte. Dans le cas de la

radiothérapie, plus la tumeur est radiosensible et plus la fenêtre thérapeutique est large (cas

2A) ; plus les tissus normaux sont radiosensibles et plus le risque est élevé d’engendrer des

dommages permanents. D’autres effets secondaires dus à la radiothérapie sont également

décrits : carcinogénicité, mutagénicité et tératogénicité. Ces effets sont assimilés comme

permanents puisque les radiations ionisantes vont induire des dommages au niveau du

génome.

- 182 -

Figure 2: La fenêtre thérapeutique : augmenter l’index thérapeutique (Bernier et al., 2004)

C- Comment réconcilier la dose tolérée avec la dose curative

La plateforme nanoXrayTM se base sur une technologie ciblant la destruction des

cellules tumorales par des nanoparticules inertes. Cette technologie ouvre de nouvelles

perspectives pour le traitement du cancer. Les nanoparticules sont conçues pour être activées

après injection par une source de rayon X externe. L’efficacité du traitement est

proportionnelle à la durée d’activation et au nombre de sessions de la radiothérapie.

La plupart des tumeurs solides possèdent des caractéristiques physiopathologiques

uniques qui ne sont pas observées dans les tissus et organes sains : angiogenèse étendue

entraînant la formation de néovaisseaux multiples présentant une architecture vasculaire

défectueuse et l’absence de drainage lymphatique. Ces caractéristiques sont responsables du

phénomène de perméabilité et de rétention de macromolécules (dit "effet EPR", pour

Enhanced Permeability and Retention effect) dans les tumeurs solides. Cette perméabilité

vasculaire spécifique des tumeurs permet l’accumulation des nanoparticules nanoXrayTM.

Tumeur: contrôle

Dommages des tissus normaux

Dose cumuléeDose cumulée

Cas non favorableCas favorable

Rép

onse

du

tissu

(%

)

Rép

onse

du

tissu

(%

)

A B

Tumeur: contrôle


Tumeur: contrôle


Dose cumuléeDose cumulée

Cas non favorableCas favorable

Rép

onse

du

tissu

(%

)

Rép

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du

tissu

(%

)

A B

- 183 -

L’utilisation des produits issus de la plateforme nanoXrayTM est destinée à résoudre le

problème majeur de la radiothérapie: la destruction des tissus cancéreux en limitant l’énergie

délivrée aux tissus sains.

La plateforme nanoPDT a été conçue en considérant les voies d’administration

traditionnelles des molécules thérapeutiques : la voie intraveineuse, orale ou intramusculaire.

La plateforme nanoPDT permet la libération de molécules photosensibilisantes au site tumoral

même pour permettre de réduire la destruction des tissus environnants et ainsi minimiser les

effets secondaires.

II-DES NANOPARTICULES ACTIVABLES POUR LE TRAITEMENT DU CANCER : UN MOYEN D’ELARGIR LA FENETRE THERAPEUTIQUE

DES APPROCHES ANTI-CANCEREUSES

2.1 Introduction

2.1.1 Nanobiotix développe des nanomatériaux ‘activables’ pour apporter des solutions cliniques aux patients

Nanobiotix est une entreprise innovante qui travaille dans le domaine de la

nanotechnologie pour le traitement du cancer. Nanobiotix met au point des nanoparticules

permettant la destruction spécifique de cellules cancéreuses par la génération de réactions

physiques contrôlées. Ces nanoparticules sont activables par différentes sources d’énergie

externes selon une utilisation de type « on » et « off ».

Pour créer ces nouveaux nanomatériaux ‘activables’, Nanobiotix a développé une

technologie fondée sur deux axes majeurs : la compréhension fine des mécanismes

biologiques et la capacité à élaborer des structures complexes à l’échelle nanométrique.

Nanobiotix développe différents programmes de recherches, basés sur le concept de

particules activables, pour élargir le champ d’application des nanoparticules en médecine :

� La plateforme nanoXrayTM est basée sur le concept de nanoparticules

cristallines activables par rayons X.

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� La plateforme nanoPDT est basée sur le concept de nanotransporteurs

encapsulant des agents photosensibilisants pour le traitement du cancer.

� La plateforme nanoMag est basée sur le concept de particules magnétiques

pour le traitement et le diagnostic du cancer.

� La plateforme « libération de médicaments actifs » est basée sur le concept

de systèmes de libération de médicaments par stimuli externes.

La plateforme nanoXrayTM développe des nanomatériaux dont les propriétés

structurales permettent d’interagir avec les rayons X, permettant d’augmenter localement les

effets de la radiothérapie. NBTXR3 représente la première génération de produits issus de

cette plateforme. NBTXR3 est destiné à être injecté par voie intratumorale chez les patients,

les nanoparticules étant préférentiellement internalisées dans les cellules cancéreuses. Les

patients sont ensuite exposés à des rayons X qui vont activer les nanoparticules et permettre

la destruction sélective des tissus tumoraux. NBTXR3 a pour ambition de lutter contre les

cancers les plus dévastateurs comme celui des poumons, du pancréas, du colon, de la prostate

ou du sein.

La plateforme nanoPDT développe des nanotransporteurs qui offrent la possibilité de

modifier de façon significative la pharmacocinétique de photosensibilisants comme la

protoporphyrine IX (Pp IX). Au-delà, et de façon toute aussi importante, ces

nanotransporteurs offrent la possibilité de contrôler la disponibilité subcellulaire. Augmenter

la biodisponibilité au niveau de la tumeur, réduire l’accumulation du photosensibilisant au

niveau de la peau et distribuer de manière différenciée le nanotransporteur au niveau des

organelles des cellules, constituent les effets majeurs apportés par les produits issus de la

plateforme nanoPDT.

Les mécanismes d’action des produits développés par Nanobiotix sont basés sur des

effets physiques et non biologiques. Cette approche permet une véritable rupture au regard

des thérapies usuellement développées. Ces matériaux possèdent en effet un haut degré de

découplage entre le cœur de la nanoparticule, qui apporte l’effet thérapeutique recherché et le

revêtement de surface de la nanoparticule qui confère à l’ensemble de la structure sa

spécificité d’action.

Un des problèmes majeurs aujourd’hui dans le traitement du cancer est de vouloir

rendre très effectifs certains traitements et ainsi exacerber leurs effets secondaires. Deux axes

peuvent permettre de résoudre ce problème : réduire la toxicité associée au traitement ou

améliorer le contrôle ou la destruction de la tumeur. Nanobiotix se propose de jouer sur les

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deux dimensions à la fois, et d’atteindre un ratio bénéfice sur risque jamais atteint jusqu’à

présent. Il s’agit clairement de briser la corrélation classique entre efficacité thérapeutique et

toxicité associée pour l’organisme. Au-delà, le principe d’intervention des nanomatériaux,

qui se situe au niveau subcellulaire pour améliorer la fenêtre thérapeutique, représente un

nouveau paradigme.

2.1.2 Interaction entre les nanomatériaux et le milieu biologique : une compréhension nécessaire pour amener les nanomatériaux en clinique

Les connaissances des interactions entre les nanomatériaux et la biologie sont

essentielles pour comprendre comment les nanomatériaux vont communiquer avec les entités

biologiques et in fine comment les nanomatériaux et la biologie vont interagir ensemble. A la

frontière entre deux mondes scientifiques – la biologie moléculaire et cellulaire et la physique

et la chimie – les chercheurs sont aux prémices de la compréhension des interactions entre les

nanomatériaux et les entités biologiques. Les résultats issus de ces recherches vont constituer

les fondements pour élaborer des ‘nanosystèmes’ plus performants permettant de développer

des interactions adaptées avec le milieu biologique pour augmenter l’efficacité et la tolérance

de ces nanomatériaux en clinique.

Les interactions entre les nanoparticules et les protéines sont considérées comme

essentielles en nanomédecine et en nanotoxicité depuis la mise en place du concept de

nanoparticules-protéines « couronnes ». Quand les nanoparticules entrent dans un milieu

biologique, les protéines présentes dans le milieu interagissent avec les particules ce qui peut

générer une modification de leurs conformations ou altérer leurs fonctions biologiques. Cette

interaction peut aussi réduire l’efficacité du traitement et/ou induire des effets secondaires. La

nature de la surface de la particule (taille, rayon de courbure, charge, fonctionnalisation)

influence le type de protéines interagissant avec les nanoparticules. L’albumine, les

immunoglobulines (essentiellement l’IgG et l’IgM) et le fibrinogène sont les protéines qui

interagissent le plus souvent avec les nanoparticules.

Afin de pouvoir délivrer les nanomatériaux au bon moment, au bon endroit et à la

bonne concentration, plusieurs défis doivent être relevés. Les nanomatériaux doivent

s’accumuler préférentiellement au niveau des tissus tumoraux et doivent pour cela être

capables de franchir les barrières biologiques comme la peau, les intestins, les muqueuses,

mais aussi de s’internaliser dans les cellules et enfin à l’échelle subcellulaire dans les

organelles. De nombreuses études ont d’hors et déjà démontré l’influence des caractéristiques

physico-chimiques des nanoparticules pour franchir ces barrières.

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La taille et la forme des nanoparticules ont un impact sur leur biodistribution et leur

pénétration cellulaire. La charge des nanoparticules permet de gérer leurs interactions avec les

membranes cellulaires et subcellulaires. Le choix d’un revêtement de surface adapté permet

de cibler de façon passive ou active la zone à traiter.

La conception des nanomatériaux, selon le principe d’intervention souhaité, doit

cependant toujours être mise en regard des effets potentiellement toxiques qui peuvent

apparaître suite à l’interaction de ces nanomatériaux avec le milieu biologique qu’ils

rencontrent.

2.1.3 Présentation du travail de thèse : participation au développement de deux types de nanoparticules pour le traitement du cancer selon deux approches différentes

Le travail réalisé au cours de cette thèse s’intéresse à l’étude de deux axes majeurs de

recherches développés par Nanobiotix: la plateforme nanoXrayTM et la plateforme nanoPDT.

Les produits issus de ces deux plateformes ont pour but de créer et de développer des

nanomatériaux activables, utilisant des sources d’énergie externes de type « on » / « off ».

Cependant, les sources d’énergies utilisées pour activer les produits sont différentes, de part

l’énergie mise en jeu (source rayons X ou laser ; Figure 3). De plus, la voie d’administration

des produits est également différente ce qui implique une approche très distincte dans la

conception des nanomatériaux issus des plateformes nanoXrayTM et nanoPDT. Ces différentes

plateformes, basées sur des approches thérapeutiques radicalement différentes, ont comme

objectif commun de permettre un contrôle locorégional de libération.

� NBTXR3, issu de la plateforme nanoXrayTM , est une suspension biocompatible de

nanoparticules cristallines et inertes d’oxyde d’hafnium (HfO2 ; ± 70 nm) dans de l’eau pour

préparation injectable (PPI). L’oxyde d’hafnium constitue le cœur thérapeutique de la

nanoparticule, seulement lorsque ses électrons sont excités par l’application des rayons X. Le

cœur est recouvert par un revêtement assurant la stabilité et la biocompatibilité des

nanoparticules. Les applications thérapeutiques de ces particules sont des cancers de type

profonds.

� Les nanoPDT sont des nanoparticules – ou nanotransporteurs – constituées de

silice, encapsulant une molécule photosensible. La taille des nanoparticules peut être ajustée

sur une large gamme, couvrant le domaine de 10 à 200 nm. Ces nanoparticules sont activables

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par une source laser. Les applications thérapeutiques de ces particules sont des cancers de

type superficiels.

Figure 3. Deux différents types de nanoparticules nanoPDT et nanoXrayTM activables par énergie électromagnétique

Les études réalisées au cours de cette thèse participent à trois axes de recherche

primordiaux, permettant de poser les bases d’un développement des produits thérapeutiques

en clinique.

1) Etudes de l’efficacité et de la toxicité in vitro

L’approche in vitro est particulièrement pertinente pour tester la toxicité, l’efficacité

des nanoparticules, l’effet de la localisation des nanoparticules à l’échelle subcellulaire sur

l’efficacité, le type de mort cellulaire induit, mais aussi pour passer au crible différents types

de cellules tumorales et normales. Plusieurs questions doivent être approchées pour

développer les nanoparticules comme « produits actifs »:

- Est-ce que les nanoparticules sont toxiques sur des lignées tumorales et/ou saines en

l’absence d’activation?

nanoXraynanoXraynanoPDTnanoPDT nanoXraynanoXraynanoPDTnanoPDT

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- Est-ce que les nanoparticules augmentent la destruction des cellules tumorales après

activation par une source externe d’énergie selon les conditions et protocoles établis?

- Quels sont les types de mort cellulaire induits par les nanoparticules ?

- Quel est le rôle de la localisation subcellulaire, et du nombre total de nanoparticules

internalisé, sur l’efficacité ?

- Est-ce que les nanoparticules induisent la même toxicité et/ou efficacité selon les

spécificités biologiques des tumeurs : leur origine (origine épithéliale ou mésenchymateuse,

structure compartimentale différente), cellules humaines saines versus cellules tumorales

malignes, caractéristiques de radiosensibilité (lignées radiosensibles ou radiorésistantes)?

2) Mécanisme d’action : trafic intracellulaire des nanoparticules

L’étude mécanistique des nanoparticules in vitro est essentielle pour la compréhension

et la mise au point des protocoles d’administration des nanoparticules et des conditions

d’activation dans des modèles in vivo. Les études de pénétration cellulaire, de localisation et

de clairance des nanoparticules sont la base de la compréhension du trafic intracellulaire.

Plusieurs axes de recherches sont explorés :

- Par quel mécanisme les nanoparticules sont-elles internalisées ?

- Quelle est la cinétique d’entrée des nanoparticules ?

- Où sont localisées les nanoparticules dans les cellules ? Dans quelles organelles ?

- Est-ce que les nanoparticules ressortent des cellules ou bien restent-elles confinées

dans des organelles pour être dégradées ?

3) Etudes de la performance et de la tolérance in vivo

Les études in vivo englobent la biodistribution des nanoparticules, l’accumulation et la

dispersion des nanoparticules dans la tumeur et leur toxicité. Les tumeurs peuvent être

apparentées à des « organes » spécifiques, différents des organes/tissus dont elles sont issues.

Ces deux entités génétiques coexistent, et ont une structure moléculaire, métabolique et un

comportement de croissance spécifiques pour chaque patient et sont fortement liées au type

tissulaire et au stade de développement du cancer. L’exploration des spécificités biologiques

des différents cellules/tissus/organes est nécessaire. Les études in vivo doivent être soutenues

par les études in vitro pour amener un produit efficace et non toxique au stade du

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développement clinique. Les axes de recherches sont nombreux et englobent les questions

suivantes :

- Quel est la nanoparticule idéale (selon sa taille, composition, forme et

fonctionnalisation) afin d’optimiser la biodistribution et/ou la pénétration cellulaire et/ou la

localisation au niveau subcellulaire ?

- Quelle est la nanoparticule idéale (selon sa taille, composition, forme et

fonctionnalisation) afin d’optimiser la biocompatibilité ?

- Les nanoparticules sont elles stables en milieu biologique ?

Le but de toutes ces approches est de montrer le réel bénéfice de ces nanoparticules

par rapport aux approches thérapeutiques actuellement développées. Concernant la plateforme

nanoXrayTM, la radiothérapie seule constitue la référence. Pour la plateforme nanoPDT, la

molécule photosensible - Pp IX libre - est la molécule de référence.

Nanobiotix travaille dans le domaine du traitement du cancer et l’élargissement de la

fenêtre thérapeutique est essentiel pour le développement de nouveaux produits en clinique.

Deux voies différentes ont été explorées par Nanobiotix : le développement de nanoparticules

thérapeutiques inertes en combinaison avec la radiothérapie pour augmenter localement l’effet

des radiations et le développement de nanoparticules à base de silice pour améliorer la

distribution de médicaments pour la thérapie photodynamique. Plus particulièrement, l’étude

des interactions entre les nanoparticules et les cellules tumorales a été explorée.

Les interactions des nanoparticules NBTXR3 avec les cellules tumorales – pénétration

cellulaire, localisation subcellulaire et évolution au cours du temps – l’impact de la

localisation des nanoparticules sur l’efficacité, l’effet dose des nanoparticules sur l’efficacité

sont tous les points abordés dans le cadre de cette étude. Le but final étant de définir des

conditions d’administration des nanoparticules NBTXR3 dans les modèles in vivo ainsi que

les modalités d’activation par rayons X.

Les interactions des particules nanoPDT avec les cellules tumorales – pénétration

cellulaire, quantité totale de particules par cellule et cinétique de clairance – ont été étudiées

dans le but d’optimiser les conditions d’activation. La quantification des radicaux libres

générés et leur localisation ont aussi été étudiées au cours de ce travail de thèse. Une

comparaison de la biodistribution des nanoPDT dans différents modèles in vivo a été

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entreprise afin de mieux comprendre le rôle du type cellulaire, du site de greffage et de la

contribution du stroma.

2.2 La thérapie nanoXrayTM permet une augmentation significative de la

dose de rayons X au niveau de la tumeur sans changer la dose appliquée

aux tissus sains

2.2.1 Nanoparticules activables par rayons X : mécanisme d’action de NBTXR3

Les radiations ionisantes interagissent avec les atomes ou les molécules dans les

cellules, en particulier avec les molécules d’eau, pour produire des radicaux libres, qui sont

responsables de dommages cellulaires non spécifiques conduisant à la mort de la cellule.

L’interaction d’un photon X avec une molécule d’eau génère l’ionisation de cette dernière,

produisant un électron d’énergie cinétique élevée et un photon d’énergie réduite. Les électrons

générés vont perdre leur énergie par interactions multiples avec le milieu environnant,

produisant des radicaux libres et de la chaleur. Ces électrons sont majoritairement

responsables des effets obtenus en radiothérapie. Les rayons X sont absorbés par l’oxyde

d’hafnium, constituant le cœur de la nanoparticule, exactement comme les radiations

ionisantes sont absorbées par les molécules d’eau. Cependant, la probabilité d’absorption d’un

photon étant proportionnelle au numéro atomique (Z) et à la densité du composé qu’il

traverse, la nanoparticule d’oxyde d’hafnium va générer les mêmes types d’effets que les

molécules d’eau, mais avec un ordre de grandeur bien supérieur. Les cellules sont ainsi

détruites spécifiquement par la production localisée et contrôlée de radicaux libres.

2.2.2 Etat de l’art : des nanoparticules d’or activables par radiation ionisante pour traiter le cancer

La question de l’augmentation de la dose, liée à la présence de matériaux possédant un

Z élevé au sein de la zone à traiter par radiothérapie a été abordé, notamment via les calculs

Monte Carlo. Ainsi, les nanoparticules d’or ont été perçues comme des systèmes prometteurs

pour le traitement de tumeurs, activées par les radiations ionisantes.

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A- Trafic intracellulaire des nanoparticules d’or

Chithrani et al., 2006, 2007 et 2009, ont largement travaillé l’influence de la taille de

nanoparticules d’or sphériques sur l’internalisation cellulaire, le transport et la localisation

subcellulaire des nanoparticules. Les nanoparticules d’or (or/citrate) ont été isolées avec des

tailles de 14, 30, 50 et 74 nm par réduction du précurseur HAuCl4 en présence d’acide

citrique jouant le rôle de réducteur, mais aussi de complexant de la surface des

nanoparticules. Les lignées cellulaires HeLa (tumeurs ovariennes), MCF-7 (tumeurs

mammaires), STO (fibroblastes) et SNB19 (tumeurs du cerveau) ont alternativement servi de

support à leurs études. Les nanoparticules d’or/citrate sont internalisées par voie

d’endocytose – récepteur médié - au sein des cellules. Les résultats suggèrent que la surface

initiale des nanoparticules d’or/citrate est modifiée par les protéines du sérum, ce qui permet

aux nanoparticules d’être internalisées par reconnaissance spécifique de ces protéines avec la

membrane cellulaire. Les nanoparticules sont systématiquement observées, sous formes de

clusters, dans des vésicules de taille de l’ordre de 500 nm dans le compartiment

cytoplasmique des cellules. Le maximum d’internalisation est observé pour des

nanoparticules présentant une taille de 50 nm. Ces résultats peuvent être expliqués par un

mécanisme compétitif qui met en jeu la notion de « wrapping time », associé aux

caractéristiques de la membrane cellulaire et de cinétique de diffusion des récepteurs sur les

sites d’internalisation.

Une étude plus spécifique de nanoparticules d’or fonctionnalisées avec de la

transferrine a montré que les nanoparticules sont internalisées par un processus chlathrine

dépendant. La question de la clairance de ces mêmes nanoparticules d’or fonctionnalisées par

de la transferrine a également été abordée sur la lignée cellulaire HeLa (Chithrani et al.,

2007). Toutes les nanoparticules d’or, indépendamment de leur taille, ressortent des cellules

mais avec des cinétiques différentes ; la cinétique de clairance étant plus rapide pour les

petites particules. Les nanoparticules apparaissent localisées dans des endosomes tardifs ou

lysosomes. Ces vésicules semblent diffuser vers la périphérie de la cellule, fusionner avec la

membrane plasmique et enfin relarguer les nanoparticules dans le milieu extérieur. La

balance entre la pénétration cellulaire et la clairance (demi-vie d’internalisation) des

nanoparticules d’or de 14, 50 et 74 nm a été établie à 0,3, 0,5 et 0,75 heures respectivement.

Bien qu’un travail considérable ait été entrepris par Chithrani et al., pour étudier

l’influence de la taille et du revêtement de surface des nanoparticules d’or sur

l’internalisation cellulaire, le transport et la localisation subcellulaire des nanoparticules,

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aucune étude n’a été publiée à ce jour – dans l’état de nos connaissances – pour évaluer

l’efficacité de ces nanoparticules par activation avec des radiations ionisantes.

La localisation des nanoparticules d’or peut être suivie grâce aux greffages de sondes

fluorescentes en surface des nanoparticules ou par visualisation directe en microscopie

électronique. La localisation des nanoparticules d’or à l’échelle subcellulaire dépend

essentiellement de la fonctionnalisation de la surface de la particule mais aussi de sa taille.

Les publications décrivent en majorité les nanoparticules d’or dans le compartiment

cytoplasmique, dans des endosomes (Rahman et al., 2009 ; Chithrani et al., 2006, 2007 et

2009). Chang et al (2008) ont cependant observé la localisation de nanoparticules d’or de 13

nm et présentant un revêtement citrate dans le réticulum endothélial et l’appareil de Golgi de

cellules tumorales B16F10. Par ailleurs, deux articles décrivent une localisation de

nanoparticules d’or dans le noyau de la cellule: des clusters de 1,4 nm pouvant interagir

avec l’ADN du noyau (Tsoli et al., 2005) et des particules de 3,7 nm fonctionnalisées avec de

l’acide 3-mercaptopropionic-polyéthylène glycol (MPA-PEG).

B- Efficacité in vitro et in vivo des nanoparticules d’or

Des tests de viabilité cellulaire ainsi que des tests de clonogénicité ont été réalisées

sur la lignée MCF-7 et sur son analogue non malin MCF-10A avec des particules d’or de

10,8 nm fonctionnalisées avec de la cysteamine (AET) ou du thioglucose (Glu). Les cellules,

traitées ou non par des nanoparticules d’or – à iso concentrations en nanoparticules d’or au

niveau cellulaire – ont reçu une dose de 10 Gray (Gy) avec un générateur de rayons X (200

KVp). Cette étude démontre que les deux types de nanoparticules d’or augmentent la

sensibilité de la radiation au niveau des cellules cancéreuses mais qu’aucun effet significatif

n’a été observé sur des cellules saines (Kong et al., 2008).

Rahman et al., 2009 ont étudié les interactions entre des nanoparticules d’or (1,9 nm)

et un modèle de cellules endothéliales (BAECs). Les cellules ont été traitées avec des

concentrations en nanoparticules d’or croissantes de 0,25 mM à 1 mM. Les études de survie

cellulaire, pour différentes doses d’irradiation (0, 1, 2, 3, 4 et 5 Gy) avec un générateur de

rayon X (80 KVp et 150 KVp) ont montré un effet dose particules dès 0,25 mM et au-delà.

Cependant une étude de viabilité cellulaire, en absence d’activation, a aussi montré une

cytotoxicité induite par la seule présence des nanoparticules, fonction de la concentration en

nanoparticules (30% de diminution de viabilité cellulaire à été observée pour une

concentration en nanoparticule de 1 mM).

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Seules deux études précliniques ont été publiées à ce jour sur l’utilisation de

nanoparticules d’or activées par des radiations ionisantes pour traiter des tumeurs. Dans une

première étude menée par Hainfeld et al. (2004), la pousse de tumeurs mammaires de type

EMT-6 implantée sur des souris a été suivie. Deux minutes après une injection intraveineuse

de particules d’or de 1,9 nm, une dose unique de 30 Gy a été délivrée par une source X (250

kVp) sur la tumeur. 86% de taux de survie à 1 an ont été obtenus pour les animaux ayant reçu

le traitement avec les nanoparticules contre seulement 20% pour les animaux exposés à la

radiothérapie seule. Dans une deuxième étude, Chang et al., 2008, ont étudié l’efficacité de

nanoparticules d’or de 13 nm sur des souris porteuses de cellules de mélanomes B16F10.

Vingt quatre heures après injection, une dose unique de 25 Gy a été délivrée par une source

d’électron de 6 MeV. Une réduction significative de la progression tumorale a été démontrée.

Les données de ces deux études précliniques sont reprises dans la figure 4 :

Taille des particules

d’or

Ratio du pic tumeur sur

tissue normal

Clairance

Temps d’irradiation

après injection i.v

Dose d’irradiation

Hainfeld 1.9 nm 3.5 / 1

à 5 min post i.v

tumeur : 41 min normal : 24 min

2 min 30 Gy

250 kVp X-ray

Chang 13 nm 6.4 / 1

à 24 heures post i.v

/ 24 heures 25 Gy

6 MeV e-

Figure 4. Comparaison des protocoles d’administration et d’activation des nanoparticules

d’or

Des nanoparticules d’or présentant des tailles supérieures à celles proposées dans ces

deux études précliniques pourraient être intéressantes pour des traitements impliquant des

protocoles d’irradiation fractionnée. En effet, la taille des nanoparticules devrait permettre

leur rétention au niveau du site tumoral (Chang et al., 2008) et favoriser leur accumulation

intracellulaire (Chithrani et al., 2006, 2007) ce qui réduirait la nécessité d’administrations

répétées. Au-delà, les deux études présentées utilisent des doses d’irradiation très élevées. Ces

doses ne sont pas adaptées pour des protocoles utilisés en clinique, surtout dans un contexte

de radiation fractionnée.

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Par ailleurs, les études de toxicité réalisées sur les nanoparticules d’or restent encore

peu nombreuses et disparates. Il semble difficile aujourd’hui de juger de la pertinence d’un

potentiel développement de ces produits en clinique.

2.2.3. Augmentation de l’effet de la radiothérapie par les nanoparticules nanoXrayTM

Article 1: « Importance de la biodisponibilité des nanoparticules

NBTXR3 dans les endosomes pour optimiser l’efficacité de la

radiothérapie sur des cellules tumorales humaines de colon »

Virginie Simon, Ping Zhang, Laurence Maggiorella, Agnès Pottier, Elsa Borghi, Laurent

Levy, Julie Marill ; en préparation

Résumé de l’article:

Le concept innovant de concevoir des nanoproduits d’oxyde métallique pour des applications

anti-tumorales marque une avancée considérable dans l'utilisation de nanomatériaux comme

produits thérapeutiques. NBTXR3 est une suspension aqueuse stérile de nanoparticules,

composée d'un cœur d’oxyde d’hafnium et d’une couche superficielle biocompatible. Le cœur

dense et inerte est la cible d’interactions avec les radiations ionisantes. L’interaction entre les

radiations ionisantes et les nanoparticules NBTXR3 produit des électrons qui vont perdre leur

énergie par interactions multiples avec le milieu environnant, produisant des radicaux libres.

Les nanoparticules NBTXR3 permettent une augmentation significative de la dose de rayons

X dans les lignées tumorales de colon HCT 116 et HT-29 et présentent un effet concentration

dépendant. L’interaction des nanoparticules NBTXR3 avec les membranes est indépendante

d’un processus spécifique d’internalisation. L’étude de l’internalisation des nanoparticules

dans les cellules, leur localisation cytoplasmique et leur efficacité a montré une corrélation

entre la localisation intracellulaire des nanoparticules et l’augmentation de la cytotoxicité des

radiations sur les cellules tumorales HCT 116. Les effets intracellulaires engendrés par les

nanoparticules NBTXR3 activées ont été observés aussi bien au niveau cytoplasmique que

nucléaire. Les nanoparticules présentent un temps de résidence long au niveau du cytoplasme

et se retrouvent dans les endosomes tardifs. La biodisponibilité spécifique des particules au

niveau des endosomes semble être le paramètre clef pour augmenter significativement la

destruction des cellules tumorales par les radiations.

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2.3 Des Pp IX nanotransporteurs créent une localisation et une

biodistribution entre la tumeur et les tissus sains différenciées:

élargissement de la fenêtre thérapeutique de la thérapie photodynamique

2.3.1 Mécanisme d’action de la thérapie photodynamique

La thérapie photodynamique est basée sur un mécanisme de photo oxydation de la

matière. Ce type de traitement requiert: une molécule photosensible qui s’accumule

préférentiellement au sein des tumeurs, une source lumineuse capable d’exciter cette

molécule et un milieu riche en oxygène. L’oxygène singulet, l’agent cytotoxique principal

produit lors de la thérapie photodynamique, est une forme hautement réactive de l’oxygène

produit à partir du dioxygène cellulaire.

Lors de l’exposition à des rayonnements laser, les molécules photosensibles génèrent

la formation de radicaux libres: les chromophores activés permettent la transformation de

l’oxygène moléculaire environnant et des espèces réactives de l’oxygène en radicaux libres,

qui sont des espèces hautement réactives provoquant des dommages irréversibles dans les

cellules tumorales. L’oxygène singulet réagit localement et rapidement : la migration d’un

oxygène singulet est inférieure à 0,02 µm et celui-ci possède un temps de demi-vie d’environ

52 µs (Rossi et al., 2008). Les effets oxydatifs induits par la thérapie photodynamique sont

ainsi confinés à une région restreinte. De plus, les longueurs d’ondes utilisées pour activer la

molécule photosensible, ne permettent pas de traverser une grande épaisseur de tissu.

NanoPDT est, de ce fait, destiné préférentiellement aux cancers superficiels comme celui de

la peau, de l’oesophage, de la vessie ou de l’estomac. Lorsque l’activation par le laser cesse,

les nanoparticules reviennent à leur état inerte.

2.3.2 Présentation des photosensibilisants avec autorisation de mise sur le marché

La famille des porphyrines a généré un grand nombre de produits actuellement sur le

marché ; Photofrin®, Levulan®, Metvix® and Visudyne® en sont les dérivés. Le médicament

Photofrin® est le médicament qui a la plus longue histoire clinique et qui a été utilisé sur le

plus grand nombre de patients. Il s’agit d’un mixte complexe d’environ 60 composants qui

combine des monomères, des dimères et des oligomères dérivant de l’hématoporphyrine. La

famille des chlorines a conduit à trois produits utilisés fréquemment en clinique : Foscan®

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(composé de temoporphyrine), LS11® (composé de talaporfine) et Photochlor® (composé de

2-devinyl-2-(1-hexyloxyethyl) pyropheophorbide : HPPH).

Enfin, la famille de texaphyrine a permis de développer le produit commercial

Antrin® qui est synthétisé à partir du lutexaphyrine.

Le premier succès du Photofrin® a été établi pour le traitement des cancers de la

vessie. La première autorisation de mise sur le marché (AMM) en 1993 par la Food and Drug

Administration (FDA) a été donnée au Canada puis en 1995 aux Etats-Unis pour le traitement

du cancer de l’œsophage. Depuis le médicament Photofrin® a obtenu des AMM dans de

nombreux pays : en Europe et aux Etats-Unis pour le cancer de l’œsophage de type Barett ; au

Japon pour des dysplasies cervicales ainsi que pour les cancers gastriques ; au Canada pour le

cancer de la vessie papillaire ; au Canada, Danemark, Finlande, France, Irlande, Japon,

Hollande, Angleterre et Etats-Unis pour le cancer de l’œsophage et au Canada, Danemark,

Finlande, France, Irlande, Japon, Hollande, Angleterre et Etats-Unis et l’Allemagne pour le

cancer endobronchéale.

2.3.3 Administration par voie générale des photosensibilisants destinés à un traitement local

A- Principales caractéristiques pharmacocinétiques du Photofrin®

Des études pharmacocinétiques du Photofrin® ont été effectuées chez la souris, le rat,

le cochon d’inde, le chien ainsi que chez l’homme et se sont avérées pour tous les modèles

relativement similaires. En général, Photofrin® est injecté chez les patients à la concentration

de 2 mg/kg. L’activation par laser (A= 630 nm) a lieu entre 24 et 48 heures après injection

avec une dose d’environ 200-300 J/cm.

B- Effets secondaires du Photofrin®

Chez la souris et le rat, des études de biodistribution ont montré une accumulation du

Photofrin® au niveau du foie, de la rate et du rein. Une quantité minime a été quantifiée au

niveau de la peau (Tronconi et al., 1995).

Chez l’homme, par opposition, Photofrin® a montré une forte photosensibilité au

niveau de la peau. Ceci est dû à une biodistribution du produit assez importante à ce niveau.

Les effets secondaires sont par conséquents lourds pour les patients puisqu’ils doivent éviter

tout contact avec la lumière naturelle pendant 4 à 6 semaines.

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2.3.4 Les nanotransporteurs

A- Etat de l’art des nanotransporteurs de silice

Au regard de la littérature, il existe deux familles de nanotransporteurs à base de silice

pour des applications anti-cancéreuses en thérapie photodynamique : celle où l’agent

photosensibilisant est greffé à la particule de silice et celle où l’agent photosensibilisant est

encapsulé.

Plusieurs équipes ont testé des nanoparticules de silice ayant un protocole de synthèse

chimique complexe. En effet, il s’agit d’établir un lien covalent entre le photosensibilisant et

la coque de silice. L’équipe d’Ohulchanskyy (2007) et celle de Brevet (2009) développent

respectivement des nanoparticules de silice où le dérivé du HPPH est greffé à la

nanoparticule de silice. Plus proche de nos applications, Rossi et son équipe (2008) et Tu et

son équipe (2009) ont synthétisé des nanoparticules de silice mésoporeuses dans lesquelles le

Pp IX a été greffé. Tu et al ont testé ces particules (110 nm) in vitro sur des cellules HeLa.

Les résultats indiquent que les nanoparticules sont efficaces. Une autre voie de synthèse

consiste à encapsuler (absence de lien covalent) dans une coque de silice les

photosensibilisants (Yan et al., 2003 ; He et al., 2009). L’équipe de Tang (2005) a par

exemple encapsulé du bleu de méthylène. Roy (2003) et son équipe ont encapsulé du HPPH et

montré une bonne efficacité du produit in vitro.

B- Nanobiotix a sélectionné un procédé de synthèse simple pour

développer des nanotransporteurs contenant le photosensibilisant Pp IX.

Article 2: « Synthèse d’un nouvel hybride polyvalent

(nanotransporteur), montrant une stabilité adaptée dans un

environnement biologique pour une utilisation en thérapie

photodynamique »

Edouard Thiénot, Matthieu Germain, Kelthoum Piejos, Virginie Simon, Audrey Darmon,

Julie Marill, Elsa Borghi, Laurent Levy, Jean-François Hochepied et Agnès Pottier ; soumis

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Un nouveau matériau hybride a été conçu par un procédé de synthèse simple, basé sur

l’approche sol-gel, pour encapsuler efficacement un photosensibilisant, le Pp IX et préserver

ainsi son activité intacte dans un environnement biologique, pour son utilisation en thérapie

photodynamique. Ces matériaux, les nanotransporteurs, ont été obtenus sous formes

sphériques avec des tailles pouvant être ajustées entre 10 nm et 200 nm. La capacité du Pp IX

encapsulé dans les nanotransporteurs à libérer efficacement les radicaux libres sous irradiation

laser a été démontrée. De même, sa capacité à induire la mort de cellules tumorales a été

testée avec succès in vitro. La stabilité des nanotransporteurs a été suivie par absorbance

ultraviolet et par émission de fluorescence à la fois en milieu aqueux et en milieu 100%

sérum. Une perte de stabilité – évaluée par la capacité de la molécule Pp IX à générer des

espèces réactives – a été observée après 2 heures de mûrissement des nanotransporteurs dans

le milieu 100% sérum. La flexibilité des nanotransporteurs a été envisagée comme un

paramètre essentiel pour préserver l’activité du Pp IX dans un environnement biologique. Un

second traitement de surface réalisé sur la première génération de nanotransporteurs, a permis

de former un composé bicouche et d’augmenter de façon significative la stabilité du

nanotransporteur dans les milieux biologiques. Cette seconde génération de nanotransporteurs

ouvre de nouvelles perspectives pour la thérapie photodynamique.

2.3.5 Interactions entre les nanoparticules de silice encapsulant le Pp IX et des systèmes biologiques

Article 3: « Des nanoparticules de silice encapsulant le

photosensibilisant Pp IX présentent des interactions spécifiques avec

des lignées cellulaires tumorales in vitro et des modèles de souris

porteuses de cancers humains in vivo”

Virginie Simon, Corinne Devaux, Audrey Darmon, Thibault Donnet, Edouard Thiénot,

Matthieu Germain, Jérôme Honnorat, Alex Duval, Agnès Pottier, Elsa Borghi, Laurent Levy

et Julie Marill ; accepté dans la revue Photochemistry Photobiology.

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Des nanoparticules de silice (nanotransporteurs) encapsulant le Pp IX, développées pour des

applications en thérapie photodynamique, ont été testées à la fois dans des modèles in vitro et

in vivo. Cette étude a pour ambition de mieux appréhender le rôle des différents facteurs

biologiques sur la génération de photodommages induits par les nanoparticules. Ces

nanoparticules de silice encapsulant le Pp IX, les nanotransporteurs de première génération,

ont montré leur efficacité dans des conditions d’activation mettant en jeu des températures

extrêmes (4°C), ce qui suggère une plus grande proportion d’internalisation par voie passive

que par voie active. Pour la première fois, la clairance cellulaire de ces nanotransporteurs a été

démontrée. L’estimation de la viabilité cellulaire a été établie dans six lignées de cellules

tumorales. Pour tous les types cellulaires, les nanotransporteurs sont plus efficaces que le Pp

IX libre. Un fort signal fluorescent démontre la colocalisation des radicaux libres générés et

des nanoparticules, corrélé à 100% de mortalité cellulaire. Des études in vivo réalisées sur des

souris porteuses des lignées HCT 116 (tumeur de colon ; cellules injectées en sous-cutané),

A549 (tumeur des poumons ; cellules injectées en sous-cutané) et de glioblastome multiforme

(tumeur du cerveau ; fragment de tumeur injecté en sous-cutané), ont montré une meilleure

accumulation des nanoparticules au niveau de la tumeur que dans le groupe contrôle

(molécule libre), soulignant leur forte sélectivité pour les tissus tumoraux. Comme observé

dans les tests in vitro, le type de tumeur s’avère être un facteur déterminant pour

l’accumulation des nanoparticules. Cependant l’environnement tumoral peut potentiellement

encore plus influencer ces différents temps d’accumulation. Ces résultats renforcent le fait que

ces nanotransporteurs peuvent devenir une nouvelle alternative pour des applications locales

de la thérapie photodynamique.

III-RESULTATS

Les nanoparticules NBTXR3, issues de la plateforme nanoXrayTM, activables par

radiothérapie, ainsi que les nanotransporteurs nanoPDT développés pour des applications en

thérapie photodynamique, ont pour ambition d’élargir la fenêtre thérapeutique. Ces deux

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produits thérapeutiques sont destinés à rendre les traitements des cancers plus efficaces et

moins nocifs pour les tissus sains.

-Recherches au sein de la plateforme nanoXrayTM

Les nanoparticules NBTXR3 induisent une importante diminution de la survie des

cellules HCT 116 après irradiation à la dose de 2 Gy. Une différence significative de viabilité

cellulaire a été mise en évidence avec les nanoparticules NBTXR3 sous irradiation aux

concentrations de 100, 200, 400 et 800 µM comparativement au contrôle (radiothérapie

seule). Un effet concentration dépendant a été observé sur la viabilité cellulaire. Des tests de

clonogénicité réalisés à la fois sur la lignée radiosensible HCT 116 et sur la lignée

radiorésistante de colon HT-29 ont clairement démontré un effet seuil de la radiodestruction

pour une concentration en nanoparticules NBTXR3 de 50 µM et 100 µM respectivement, par

rapport à la radiothérapie seule. Aucun signe de toxicité associé aux nanoparticules NBTXR3

n’a été observé sur ces deux types de lignées cellulaires.

Concernant les études réalisées sur une lignée de tissu sain (lignée de fibroblastes

transformés), aucun effet différentiel n’a été observé entre les nanoparticules NBTXR3

irradiées et la radiothérapie seule à la dose de 2 Gy (résultats non montrés).

La viabilité cellulaire a été mesurée pour différents temps d’incubation avec les

nanoparticules NBTXR3 en présence des cellules HCT 116. L’efficacité dépend du temps

d’incubation. Elle est observée dès 2 heures. Une différence significative est observée entre 2

heures et 4, 15 et 24 heures d’incubation.

Les images en microscopie électronique à transmission (MET) des cellules HCT 116

incubées 2 et 24 heures avec les nanoparticules NBTXR3 présentent une internalisation

différenciée. Après 2 heures, 20% des cellules ont internalisé les nanoparticules NBTXR3.

Après 24 heures, 80% des cellules ont internalisé les nanoparticules NBTXR3. Ces dernières

sont localisées dans le compartiment cytoplasmique au sein d’endosomes. Pour un nombre

total de nanoparticules NBTXR3 internalisées par cellules (entre 60±7,7 mg.kg-1 et 103±3,5

mg.kg-1 de nanoparticules NBTXR3 quantifiées pour 100 000 cellules sur la lignée HCT 116),

la localisation des particules dans les endosomes apparaît être le paramètre essentiel pour

augmenter de façon significative l’effet de la radiothérapie.

La viabilité cellulaire est similaire à celle observée pour la radiothérapie seule pour des

cellules traitées 2 heures avec NBTXR3 et irradiées immédiatement après avoir renouvelé le

milieu. A l’inverse, la viabilité cellulaire diminue de façon significative, par rapport à la

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radiothérapie seule, pour des cellules traitées 2 heures avec NBTXR3 et irradiées 48 heures

après avoir renouvelé le milieu. Parallèlement, la quantification des nanoparticules NBTXR3

au niveau cellulaire, montre une même concentration de nanoparticules pour des cellules

incubées 2 heures avec NBTXR3, suivie par un changement du milieu et incubées aux temps

supplémentaires de 0 et 48 heures. Ces résultats renforcent l’hypothèse de la nécessité d’une

localisation intracellulaire des nanoparticules pour produire un effet thérapeutique sous

irradiation et suggèrent l’absence du phénomène de clairance à une échelle de temps d’au

moins 48 heures.

L’analyse et l’interprétation des images MET des cellules HCT 116 traitées avec

NBTXR3 et irradiées suggèrent une mort cellulaire par apoptose et par autophagie. Après

irradiation, les cellules traitées par NBTXR3 montrent une proportion significative de

micronoyaux par rapport à la radiothérapie seule. Une désorganisation cellulaire est aussi

observée ainsi que la fusion d’endosomes contenant les nanoparticules pour former des

vésicules de grandes tailles, concentrées dans une zone du cytoplasme. La fusion

d’endosomes a aussi été décrite pour des temps prolongés d’incubation.

Les nanoparticules NBTXR3 semblent interagir de façon non spécifique avec les

membranes cellulaires des cellules HCT 116 et pénétrer par endocytose. L’observation des

cellules incubées 24 heures avec les nanoparticules, 96 heures après irradiation, montre

encore la présence des nanoparticules dans des vésicules (endosomes et lysosomes). Les

nanoparticules semblent rester dans le cytoplasme.

En parallèle du travail réalisé au cours de cette thèse, les nanoparticules NBTXR3 ont

été injectées par voie intratumorale sur des souris porteuses de tumeurs HCT 116 greffées sur

le flanc. Une irradiation locale de la tumeur a été effectuée. Une régression, voire une

éradication, tumorale a été observée chez tous les animaux irradiés alors que le groupe

radiothérapie seule ne présente qu’un retard de croissance tumorale : après 60 jours, 90% des

animaux ne portaient plus de tumeurs pour le groupe traité avec les nanoparticules NBTXR3

et irradié. Le groupe avec la radiothérapie seule a montré une reprise de la pousse tumorale

entraînant le sacrifice des animaux avant la fin de l’étude. Une étude de tolérance sur des

souris nudes porteuses de tumeurs HCT 116 a été effectuée par administrations répétées par

voie intraveineuse de nanoparticules NBTXR3. La dose administrée a été bien tolérée chez

l’ensemble des animaux.

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-Recherches au sein de la plateforme nanoPDT

Les nanoPDT ou nanotransporteurs sont des nanoparticules à base de silice

encapsulant la molécule photosensible Pp IX. Ces nanoparticules sont de morphologies

sphériques et monodisperses en taille. Un contrôle de leur taille entre 10 et 200 nm a été

effectué grâce à l’ajustement de la température de la synthèse entre 18°C et 37°C. L’effet de

la taille de ces nanotransporteurs de première génération a été évalué in vitro sur la lignée

HCT 116. Des tests de viabilité cellulaire ont montré des résultats équivalents quelque soit

leur taille en accord avec les résultats de cinétique d’internalisation et du nombre total de

particules internalisées.

Afin de comprendre les mécanismes d’internalisation de ces nanotransporteurs dans

les cellules HCT 116, des tests de viabilité cellulaire ont été effectués à 4°C et à 37°C. La

température de 4°C est connue pour bloquer la voie d’internalisation

d’endocytose/pinocytose. Les cellules HCT 116 traitées pendant 3 heures avec des

nanoparticules à 4°C et activées par laser juste après montrent une EC50 (dose efficace 50%)

équivalente à celle obtenue dans les mêmes conditions à 37°C. Cependant, le nombre total de

nanoparticules internalisées est plus faible à 4°C qu’à 37°C (résultats non montrés). Ces

résultats suggèrent une balance d’internalisation entre la voie passive et active. Les

nanoparticules sont internalisées dans le cytoplasme. Le signal fluorescent des nanoPDT est

diffus à travers tout l’espace cytoplasmique. Des études complémentaires de colocalisation de

nanoparticules permettraient de savoir plus précisément avec quelles organelles les particules

interagissent.

La question de la clairance des nanoparticules a été abordée. Les cellules HCT 116 ont

été traitées pendant 3 heures à plusieurs concentrations de nanoPDT. Puis le milieu a été retiré

et du nouveau milieu sans nanoparticule a été ajouté. Vingt quatre heures après la fin de

l’incubation, 100% des nanoparticules sont ressorties des cellules. De plus, la clairance des

nanoparticules commence très rapidement (dès 2 heures). Ces résultats montrent que

l’efficacité des nanoparticules est corrélée à la localisation intracellulaire des nanoPDT.

Un effet temps d’incubation des nanoparticules sur HCT 116 a aussi été démontré en

viabilité cellulaire. Trois heures d’incubation (avec une concentration en particules de 1 µM)

sont suffisants pour induire 100% de mort cellulaire. Le meilleur temps d’irradiation a été

défini comme étant celui de 20 min. Les conditions optimisées retenues sont: 3 heures

d’incubation, 20 min d’irradiation et 48 heures de post incubation.

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Aucun test in vitro n’a montré un bénéfice de la multi-irradiation en terme de réponse

cellulaire (résultats non montrés). Les tests de multi-irradiation pourront cependant être

entrepris in vivo afin de mettre en évidence un éventuel bénéfice.

Des tests de viabilité cellulaire et de toxicité ont été entrepris dans six lignées

différentes: HCT 116, HT-29, A431 (tumeur épidermoïde), LLBC37 (lymphoblastoïde),

MCF-7 et MDA-MB-231 (tumeurs mammaires). Pour toutes ces lignées, les nanoparticules

nanoPDT ont une efficacité supérieure (différence de plus d’un log) à celle du Pp IX libre.

Les valeurs d’EC50 des nanoparticules sont comprises entre 0,4 et 1,2 µM alors que les

valeurs d’EC50 du Pp IX libre sont comprises entre 3,4 et 32,7 µM pour l’ensemble des

lignées testées.

Une corrélation a été mise en évidence entre la génération de radicaux libres et les

concentrations en nanoPDT dans les cellules HCT 116 et HT-29. Cependant, la quantité de

nanoparticules internalisées dans la lignée HT-29 est plus faible que celle quantifiée dans la

lignée HCT 116. Un maximum de 600 fmol de nanoparticules pour 1000 cellules (3 heures

d’incubation à 5 µM de nanoPDT) a été quantifié dans la lignée HCT 116 alors qu’un

maximum de 200 fmol de nanoparticules pour 1000 cellules a été quantifié pour HT-29 dans

les mêmes conditions. Ces résultats démontrent un différentiel d’internalisation entre les deux

lignées testées qui apparaît corrélé aux valeurs respectives des EC50 observées.

Une mort cellulaire par apoptose est majoritairement décrite dans la littérature après

injection du Pp IX ou du Photofrin® activés. Le mécanisme de mort cellulaire dans toutes les

lignées testées devrait être abordé avec nos nanoparticules.

De plus, des études de biodistribution ont été réalisées sur des souris porteuses des

différentes lignées (HCT 116, A549 et glioblastome). Les résultats montrent une très bonne

accumulation de ces particules au niveau de la tumeur avec des temps d’accumulation

maximaux variables selon les modèles testés.

Dans le but d’optimiser la performance des nanoPDT, des études de stabilité dans le

milieu biologique ont été entreprises. Les résultats montrent que ces nanotransporteurs de

première génération présentent une perte de leur stabilité dès 2 heures d’incubation dans du

sérum de souris. En effet, ces nanoparticules présentent une modification du spectre

d’absorption du Pp IX et une perte d’émission de fluorescence. Afin de générer un

nanotransporteur stable dans les milieux biologiques, des nanotransporteurs de deuxième

génération ont été développés.

La seconde génération de produits nanoPDT, a été conçue en réalisant un second

revêtement de surface biocompatible (addition de sodium trimétaphosphate : STMP)

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permettant de générer un nanotransporteur bicouche. Ce nanotransporteur a montré une

excellente amélioration de la stabilité en milieu biologique – capacité du Pp IX encapsulé à

générer des radicaux libres responsables de la destruction cellulaire - jusqu’à 12 heures de

mûrissement des nanoparticules dans du sérum.

IV-CONCLUSIONS ET PERSPECTIVES

GENERALES

Ce travail a permis d’enrichir les connaissances dans le domaine novateur de la

nanomédecine. Spécifiquement, l'étude des interactions entre les nanoparticules activées par

différentes sources d'énergie électromagnétiques externes et des cellules cancéreuses pour

élargir la fenêtre thérapeutique a été explorée.

La nanotechnologie permet de générer des nanomatériaux présentant de nouvelles

fonctions, comme l’utilisation de nanoparticules activables pour traiter le cancer. Elle permet

aussi de générer des nanomatériaux comme vecteurs de molécules thérapeutiques pour

modifier la pharmacocinétique du principe actif et la biodisponibilité subcellulaire. La taille

nanométrique des objets permet d’agir à l’échelle du monde biologique. Cependant il apparaît

essentiel aujourd’hui que ces objets interagissent avec les systèmes biologiques pour être

utilisés sans danger et de façon effective en médecine. Quand le monde des nanoparticules

rencontre celui de la médecine, de nouvelles modalités pour le traitement du cancer sont alors

envisagées, comme celle de l'utilisation d’une source énergétique externe à l’organisme pour

activer les médicaments.

Ce travail de doctorat a contribué au développement de deux types de nanoparticules,

NBTXR3 et nanoPDT, destinées à être utilisées pour une même application, le cancer, par

deux approches différentes.

NBTXR3 est le produit phare de plateforme nanoXrayTM. Nous avons mis en évidence

dans des lignées tumorales que les nanoparticules interagissent de façon non spécifique avec

la membrane cellulaire et pénètrent dans les cellules via un mécanisme d’endocytose, que la

localisation de ces nanoparticules est cytoplasmique via leur confinement dans des endosomes

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puis dans des lysosomes. Les nanoparticules résident dans les endosomes et dans les

lysosomes. De plus, l’importance de l’internalisation de ces nanoparticules au niveau

cytoplasmique apparaît nécessaire pour obtenir une augmentation significative de l’effet de la

radiothérapie.

L’ensemble des ces résultats participe à l’établissement du rationnel de développement

de NBTXR3. L’étude de l’efficacité des nanoparticules activées par différents types d’énergie

et selon les spécificités biologiques des tumeurs, constitue des axes de recherches qui doivent

être explorés.

Des études précliniques se poursuivent actuellement. Des résultats préliminaires

d'efficacité in vivo montrent que l’administration de NBTXR3 à des souris porteuses de

tumeurs de colon (HCT 116) xénogreffées permet une régression totale de tumeur et ce pour

tous les animaux traités. La plateforme nanoXrayTM développe un traitement totalement

novateur qui pourra être utilisé seul ou en synergie avec les protocoles de traitements déjà

existants: la chimiothérapie, la chirurgie, l’immunothérapie, etc.

La plateforme nanoPDT axe sa technologie sur l’utilisation de nanoparticules comme

nanotransporteurs. Nous avons prouvé que les nanoparticules de silice encapsulant le Pp IX

étaient des transporteurs efficaces pour délivrer des médicaments mais aussi pour les protéger

de l'exposition à un milieu aqueux environnant. Ces nanotransporteurs permettent de piéger

efficacement le Pp IX et présentent une gamme de porosité appropriée pour libérer les

radicaux libres après activation par laser dans le milieu environnant. L’encapsulation

physique du Pp IX s’avère être la voie de synthèse la plus simple possible. L’addition d’un

second composé (le STMP) à la surface des nanoPDT, pour former un système bicouche,

permet leur stabilité dans un milieu avec sérum.

De plus, des études précliniques ont démontré la non toxicité de ces nanotransporteurs

que ce soit in vitro ou in vivo. Nous avons montré que les nanoparticules de 10 à 60 nm

avaient le même comportement tant au niveau de l’internalisation que de l’efficacité

cellulaire. Une internalisation en majorité par voie passive a été démontrée dans la lignée

HCT 116. Pour l’ensemble des six lignées cellulaires testées, la localisation des

nanoparticules est la même à savoir diffuse dans tout le compartiment cytoplasmique. Une

internalisation optimale des nanoPDT dans les cellules entraîne par conséquent un maximum

de libération de radicaux libres générés et ainsi induit une réponse optimale en terme de mort

cellulaire. Pour toutes les lignées cellulaires testées, les nanotransporteurs nanoPDT sont plus

efficaces que la molécule de référence.

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Des études in vivo réalisées sur des souris porteuses de tumeur ont permis de réaliser

les premières observations de l’accumulation du produit en terme de ratio tumeur/peau. Les

résultats montrent une forte accumulation du produit dans la tumeur. De plus, l'analyse semi

quantitative réalisée sur trois modèles différents de tumeurs a montré des différences de

cinétique d’accumulation des nanoPDT.

Récemment, Nanobiotix a publié des résultats préliminaires d’études précliniques sur

les produits issus de la plateforme nanoPDT et a validé leur utilisation dans le traitement du

glioblastome, un des cancers les plus mortels et les plus fréquents au niveau du cerveau.

Il reste bien évidemment encore beaucoup d’études à effectuer mais ces premiers

résultats précliniques nous rendent confiants quant au développement de cette plateforme

technologique prometteuse nanoPDT.

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